Nanoparticulate Photoluminescent Probes for Bioimaging

Int. J. Mol. Sci. 2022, 23, 4949. https://doi.org/10.3390/ijms23094949 www.mdpi.com/journal/ijms

Review

Nanoparticulate Photoluminescent Probes for Bioimaging:

Small Molecules and Polymers

Sanghyuck Lee 1,†, Chul Soon Park 2,† and Hyeonseok Yoon 1,3,*

1 Department of Polymer Engineering, Graduate School, Chonnam National University, 77 Yongbong-ro, Buk-gu,

Gwangju 61186, Korea; [email protected] 2 Drug Manufacturing Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF),

Daegu 41061, Korea; [email protected] 3 School of Polymer Science and Engineering, Chonnam National University, 77 Yongbong-ro, Buk-gu,

Gwangju 61186, Korea

* Correspondence: [email protected]; Tel.: +82-62-530-1778

† These authors contributed equally to this work.

Abstract: Recent interest in research on photoluminescent molecules due to their unique properties

has played an important role in advancing the bioimaging field. In particular, small molecules and

organic dots as probes have great potential for the achievement of bioimaging because of their desira-

ble properties. In this review, we provide an introduction of probes consisting of fluorescent small

molecules and polymers that emit light across the ultraviolet and near-infrared wavelength ranges,

along with a brief summary of the most recent techniques for bioimaging. Since photoluminescence

probes emitting light in different ranges have different goals and targets, their respective strategies

also differ. Diverse and novel strategies using photoluminescence probes against targets have gradu-

ally been introduced in the related literature. Among recent papers (published within the last 5 years)

on the topic, we here concentrate on the photophysical properties and strategies for the design of mo-

lecular probes, with key examples of in vivo photoluminescence research for practical applications.

More in-depth studies on these probes will provide key insights into how to control the molecular

structure and size/shape of organic probes for expanded bioimaging research and applications.

Keywords: photoluminescence; bioimaging; polymer dots; near-infrared; quantum dots; small-molecule

probes; in vivo

1. Introduction

Biological imaging technology is based on various strategies for analyzing and solv-

ing the mysteries of the human body, understanding the roots of disease, and achieving

individualized or personalized medicine. Therefore, bioimaging requires probes with

very high specificity and sensitivity. Accordingly, the exploration of materials with excel-

lent photoluminescence properties and suitable strategies for their synthesis and applica-

tion is needed to analyze the biological microenvironment and associated processes. Re-

cent non-invasive photoluminescence imaging techniques appear to represent simple

technology; however, these techniques are in fact highly versatile with real-time monitor-

ing capabilities, providing unprecedented spatial and temporal resolution. Although nu-

merous studies proposing novel imaging strategies have been published recently, further

development of phosphors with higher brightness and luminous properties is necessary.

In addition, the recent tremendous advancement of clinical technology and instruments

for photoluminescence imaging induction surgery in patients has led to the need for fur-

ther photoluminescence development, especially for the development of radioactive and

biocompatible fluorophores [1–3].

Citation: Lee, S.; Park, C.S.; Yoon, H.

Nanoparticulate Photoluminescent

Probes for Bioimaging: Small

Molecules and Polymers. Int. J. Mol.

Sci. 2022, 23, 4949.

https://doi.org/10.3390/ijms23094949

Academic Editor: Ludmilla A.

Morozova-Roche

Received: 25 March 2022

Accepted: 27 April 2022

Published: 29 April 2022

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Copyright: © 2022 by the authors. Li-

censee MDPI, Basel, Switzerland.

This article is an open access article

distributed under the terms and con-

ditions of the Creative Commons At-

tribution (CC BY) license (https://cre-

ativecommons.org/licenses/by/4.0/).

Int. J. Mol. Sci. 2022, 23, 4949 2 of 52

Photoluminescence imaging technology has specific advantages for biomedical imaging

applications, including high sensitivity, low cost, and high spatial/temporal resolution. Stud-

ies have been conducted on the use of various materials such as small-molecule luminophores

[4–6], polymer dots (Pdots) [7–9], single-walled carbon nanotubes [10–14], semiconductor

quantum dots (QDs) [15–23], and rare Earth-doped nanoparticles [24–26] as fluorescent

probes. However, each of these probes has limitations such as a low quantum yield, low sol-

ubility, high cytotoxicity, and low photostability. One of the main areas of focus in the field of

fluorescence-based bioimaging applications is far-infrared and near-infrared (NIR) fluores-

cence (650–1000 nm). Fluorescence emitted from the NIR range has recently been used in clin-

ical imaging-guided cancer surgery, demonstrating excellent potential for improvement in re-

sults [27–30]. However, the limited selection of available photoluminescence probes is an im-

portant obstacle in applying photoluminescence imaging to a wide range of biological sam-

ples. Most biological species such as water, lipids, melanin, oxyhemoglobin, and deoxyhemo-

globin absorb very little light and exhibit low scattering within the NIR region [31]. In addi-

tion, since biological tissues have very low autofluorescence within the NIR region, the use of

photoluminescence probes emitting in this region may result in high signal-to-noise contrast.

More importantly, visible light can penetrate only a few micrometers within the tissue,

whereas NIR rays (700–900 nm) can penetrate up to a few millimeters through the blood or

tissue, providing a clear surgical guide with minimal tissue damage [32,33].

Despite these achievements, more advanced bioimaging techniques are needed to fur-

ther increase the properties of absorption, scattering, and autofluorescence transmittance in

biological tissues. Recently, infrared spectral windows were classified into NIR-I (700–1000

nm) and NIR-II (1000–1700 nm) [31,34]. Images using photoluminescence probes emitting in

higher-wavelength bands can reduce autofluorescence and photon attenuation and obtain a

higher signal-to-background ratio (SBR); however, the low quantum yield and low biocom-

patibility remain important obstacles to their clinical application [35–37]. In addition, although

many inorganic and carbon nanomaterials (e.g., single-walled carbon nanotubes, QDs, and

rare Earth-doped nanoparticles) have been developed as NIR-II probes, their immunoadsorp-

tion, biocompatibility, and elimination after use remain important issues to be improved. Con-

tinuous development and potential application of NIR probes in the real-time imaging of bio-

logical processes and analytes in vivo will provide mechanistic insights into the underlying

biology and disease pathology, leading to clinical translation to ultimately realize overall im-

provements in disease diagnosis and treatment patterns.

In this review, we introduce recent studies on synthesized small molecules and semicon-

ducting polymer-based Pdots as photoluminescent probes for bioimaging. We compare the

advantages and disadvantages of these small-molecule and polymeric probes according to

their characteristics with respect to their application potential. We also provide a brief over-

view of studies using carbon dots (CDs), graphene quantum dots (GQDs), metal-organic

frameworks (MOFs), and covalent organic frameworks (COFs). We hope that this review will

help researchers select the most suitable bioimaging probe material for future studies, and we

further discuss some challenges and exciting opportunities in this emerging field.

2. Overview of Several Photoluminescent Probes

2.1. Small-Molecule Probes

Optical imaging based on small-molecule photoluminescence probes has attracted

substantial attention owing to its characteristic advantages. In particular, these probes can

help to visualize non-invasive and diverse biological components, enabling the real-time

monitoring of an organ’s dynamic processes at the molecular, cellular, and living organ-

ism levels. Above all, optical imaging using small-molecule photoluminescence probes is

a fast, sensitive, specific, and multiplexed method to detect analytes [38–42]. However,

optical imaging is generally constrained by the electromagnetic spectrum region used for

biological sampling. In the optical window shown in Figure 1a, the light absorption/emis-

sion ranges of the skin/fat and oxidized/deoxygenated whole blood are particularly low

Int. J. Mol. Sci. 2022, 23, 4949 3 of 52

at 650–950 nm and 1000–1350 nm, referred to as the first and second windows, respectively.

As relatively high light transmittance is guaranteed in these windows, photoluminescence

probes in the NIR region can provide deeper penetration and higher image quality than

photoluminescence probes emitting in the visible-light region [31,43]. To accurately detect

and monitor significant biomolecules and molecular events in real time, numerous reason-

ably designed NIR-I fluorescent probes have recently been developed, which have also been

used to aid surgical imaging treatments such as the removal of tumor tissue and photother-

mal therapy. Optical wavelengths in regions above 1000 nm further minimize the number

of photons absorbed or scattered by biomolecules and media, providing deeper penetration

and good spatial resolution with lower non-target auto-fluorescence.

Figure 1. (a) Two optical windows showing the high transparency in biological tissues and fluids.

Reprinted with permission from Ref. [31]. Copyright 2009, Springer Nature. (b) Photographs of ex-

perimental results in tissue penetration. Reprinted with permission from Ref. [43]. Copyright 2019,

Springer Nature. (c) Spectral ranges for visible light, near-infrared (NIR)-I, and NIR-II showing the

corresponding wavelengths.

Several valuable strategies have been used for manufacturing meaningful bioimaging

devices. As a typical bioimaging study, in 2018, our group [44] also highlighted the importance

of optically detecting alkaline phosphatase (ALP) with NIR small-molecule probes (Figure 2).

As schematically illustrated in Figure 2a, two different NIR fluorescent probes were synthe-

sized, consisting of a phenolic dihydroxanthene fluorophore core and a phosphate with a sul-

fonate group to improve water solubility. Although these NIR probes are almost completely

nonfluorescent, they emit fluorescence upon ALP-dependent catalytic dephosphorylation,

leading to a concentration- and time-dependent “turn-on” fluorescence mechanism (Figure

2b). The ALP level in the blood can be used to diagnose several conditions, including bone

disorders and liver dysfunction. As displayed in Figure 2c, in vivo monitoring of ALP signal

changes was successfully achieved with an NIR probe-labeled calcium-deficient hydroxyap-

atite (CHDA) scaffold implanted into mice, indicating that the NIR probe can serve as an in-

dicator of osteoblast activity during early osteoblast differentiation.

Recent studies have achieved clearer photoluminescence imaging with an improved

SBR in the NIR-II region (1000–1700 nm) than in the NIR-I region because light scattering

is reduced further in the former than in the latter [45–48]. In this vein, the focus of recent

related studies has been to develop new fluorescent probes that absorb or emit in the NIR-

II region for optical imaging. Small molecule-based fluorescent probes have the well-

known advantages of a core structure and the ability for specific modifications to target a

Int. J. Mol. Sci. 2022, 23, 4949 4 of 52

biomarker according to their physical and optical properties. Therefore, it is easy to design

a specific structure and to establish the optimal strategy for bioimaging with NIR-II fluo-

rescent reagents. Consequently, the development of small molecule-based organic fluor-

ophores for the NIR-II region is a major research hotspot in the field of biological imaging

and for the exploration of associated chemical approaches.

Figure 2. (a) Schematic illustration of the fluorescence “turn-on” process of near-infrared (NIR)

probes triggered by alkaline phosphatase (ALP). (b) Ultraviolet-visible absorption of NIR probes (5

μM) and (c) fluorescence emission spectra and fluorescence images (excitation = 685 nm) of NIR

probes (5 μM) with different concentrations of ALP (0–10−5 U mL−1; the reaction time for all concen-

trations was 1.5 min) in Tris-HCl buffer (10 mM, pH 7.4). (d) Fluorescence emission spectra of NIR-

Phos-1 (5 μM). (e) Fluorescence emission spectra of NIR-Phos-1 (5 μM) with ALP (0.1 U mL−1) over

different time periods (0–30 min) at room temperature. The spectra were obtained at 0.5 min inter-

vals. (f) Fluorescence images of NIR-Phos-1-labeled CDHA scaffolds implanted in mice. Reprinted

with permission from Ref. [44]. Copyright 2018, Elsevier.

2.2. Pdots

Recently, Pdots, which have semiconductor characteristics, have emerged as power-

ful probes in various applications such as bioimaging/biosensing and photodynamic ther-

apy [8,49,50]. Compared with conventional photoluminescence probes, rationally de-

signed Pdots may exhibit high single-particle brightness, a high radiative rate, and out-

standing photostability. The fluorescence of Pdots is derived from the exciton transition

from the singlet state to the ground state, and phosphorescence is accomplished by the

exciton transition from the triplet state to the ground state. By contrast, thermally acti-

vated delayed fluorescence (TADF) is a distinct mechanism in which the exciton transition

from the triplet excited state to the singlet excited state occurs through a thermally

Int. J. Mol. Sci. 2022, 23, 4949 5 of 52

activated reverse inter-system crossing process. TADF consists of both short (nanosecond

scale) and long (micro- to millisecond scale) emission lifetime regimes, facilitating its use in

time-resolved fluorescence imaging [51–55]. Because TADF takes advantage of the long-lived

triplet excited states that are more vulnerable to luminescence quenching, the TADF process

should be protected from undesirable quenching mechanisms, making it more useful for bio-

logical imaging in practice. One method to protect against quenching is to encapsulate TADF

fluorophores within water-soluble nanoparticles; however, over time, the fluorophores are re-

leased from the nanoparticles, thereby reducing the fluorescence signal’s intensity. A typical

solution to overcome this limitation is to use a semiconducting polymer with an intrinsic

TADF property, such as hydrophilic polymers, for producing a nanoparticulate polymer

probe. Similarly, other mechanisms of action such as Förster resonance energy transfer, pho-

toinduced electron transfer, aggregation and disaggregation-induced emission, intramolecu-

lar charge transfer (ICT), and motion-induced changes in emission can be more efficiently re-

alized within polymer matrices. Furthermore, the surface of Pdots can be modified to enhance

their suitability for bioimaging. For example, Pdots generally consist of hydrophobic π-conju-

gated polymers. However, when mixed with a hydrophilic polymer such as polyethylene gly-

col (PEG), Pdots can minimize non-specific binding with non-target species. This versatile sur-

face functionalization ability of Pdots further provides various opportunities for their applica-

tions, such as in sensors, cell labeling, and bioimaging. Moreover, the appropriate design and

strategy for the surface functionalization of Pdots can facilitate binding to specific molecules

covalently when targeting tumor cells and stem cells. Recently, sensing platforms have been

designed for detecting chemical ions and for molecular recognition in point-of-care diagnostic

devices based on Pdots, showing high sensitivity and accuracy [56–58]. Due to their charac-

teristics of improved luminosity and high luminescence brightness, Pdots can also be used in

selective cell targeting to provide a reverse tile platform for theragnostics. Therefore, many

recent studies have utilized Pdots to discover applications for intracellular single-particle

tracking, lateral flow or blot-style analysis, and immuno-labeling of cancer cells [59–65].

2.3. CDs and GQDs

Due to their brightness and photostability properties, inorganic semiconductor QDs

have attracted substantial attention as a potential alternative to organic fluorescent probes

in bioimaging. However, their drawbacks, including poor water solubility, blinking, and

intrinsic toxicity, pose challenges for the use of QDs in bioimaging applications [66–69],

leading to further research attempting to enhance their properties.

Since the discovery of luminescent CDs in 2004 [70], extremely diverse investigations

have been conducted on synthetic methods, the exploration of reaction mechanisms, and

their potential applications due to their remarkable characteristics such as tunable optical

properties, good photostability, and superb biocompatibility [71–75]. In particular, CDs

not only have surface functional groups enabling post-synthetic functionalization but also

have high biocompatibility, offering great potential in applications such as the real-time

tracking of drugs, tumor therapy, and bioimaging/biosensing. Furthermore, the methods

of preparing CDs are mostly simple, cost-effective, and environmentally friendly. CDs can

be prepared in one-step procedures, using microwave and hydrothermal methods, and

with almost any type of materials (e.g., amino acids, glucose, dopamine, and citric acid)

as precursors, including those obtained from natural sources. Recently, diverse, effective

methods for manufacturing photoluminescent CDs have been developed, which can be

divided into top-down and bottom-up approaches [76–79]. Laser ablation, chemical oxi-

dation, electrochemical oxidation, and arc discharge methods are examples of top-down

methods, whereas microwave irradiation, thermal decomposition, hydrothermal treat-

ment, and plasma treatment are examples of bottom-up methods. By researching further

developments of these various methods, the applications of CDs can be expanded, with

remarkable results expected in the future.

GQDs consist of nanometer-sized fragments of graphene layers with chemical func-

tional groups. Graphene has a hexagonal lattice and consists of two-dimensional sheets

Int. J. Mol. Sci. 2022, 23, 4949 6 of 52

[80]. Furthermore, the highly ordered and closely carbon atom-packed structure grants gra-

phene unique and useful properties [81,82]. One such property is the zero-energy bandgap

due to the overlapping of graphene’s valence and conduction bands. However, the zero

bandgap is attributed only to immaculate graphene, which refers to an infinite-dimension

graphene with no defects. By contrast, actual graphene contains defects with a finite value

in physical dimensions, resulting in a non-zero bandgap. Consequently, GQDs consisting

of nanoscale fragments of graphene exhibit quantum confinement effects with a tunable

bandgap [83–86]. Diverse research has revealed that photoluminescence excitation and

emission wavelengths can be changed by adjusting the size of GQDs, changing the surface

properties or introducing dopants such as boron and nitrogen atoms into the carbon lattice

of graphene. The photoluminescence characteristics of GQDs can be adjusted to improve

their suitability for optical applications and effectiveness in bioimaging/biosensing. In addi-

tion to a dopant within the lattice, hydroxyl and carboxyl groups, which are oxygen-rich

functional groups, exist on the edge of GQDs. These functional groups enhance the water

solubility, thereby facilitating the biological applications of GQDs.

2.4. MOFs and COFs

MOFs consist of organic ligands and metal ions (or clusters). Since MOFs have re-

markable advantages such as a large surface area, high porosity, and tunable chemical

composition, they have shown strong potential for various sensing, catalytic, and biolog-

ical applications [87–89]. In particular, many recent studies demonstrated the use of MOFs

with photoluminescence properties in bioimaging/biosensing and disease treatment [90].

The organic ligands of MOFs directly influence their luminescent characteristics. In addi-

tion, for biological applications, the organic ligands of MOFs have to be carefully selected

by considering the biocompatibility and toxicity. Furthermore, the fluorescence range of

MOFs can be expanded by combination with other luminescent species.

COFs have been continuously studied since their discovery in 2005 [91]. COFs feature

organic porous networks that are covalently linked with organic materials. In contrast to

MOFs, COFs consist of light elements such as hydrogen, boron, carbon, nitrogen, and ox-

ygen. Furthermore, in contrast to amorphous organic polymers, COFs have a well-defined

two- or three-dimensional crystalline structure. COFs have attracted substantial research

attention due to their beneficial characteristics such as a tunable chemical structure/func-

tionality, porosity, high crystallinity, and large surface area. Consequently, COFs show

strong potential for applications in optoelectronics, such as in catalysts, energy stor-

age/conversion devices, and in sensors capable of detecting explosives, ammonia, chiral

species, small molecules, and DNA [92–102]. In general, the π-conjugated skeleton en-

dows COFs with a unique fluorescence property. Most studies on COFs have mainly fo-

cused on constructing conjugated structures using different chemical units or establishing

different types of bonds between them.

Table 1 summarizes the advantages and disadvantages of the above-mentioned photo-

luminescent probes. The following sections address the characteristics of the small-molecule

and polymeric probes for bioimaging applications, with remarkable recent research examples.

Int. J. Mol. Sci. 2022, 23, 4949 7 of 52

Table 1. Summary of the probes currently used in bioimaging.

Probe

Type Advantage Disadvantage Strategy Reference

Small

molecule

- Well-defined chemical

structure

- Structural tunability

- Low cytotoxicity

- High cell permeability

- Rapid metabolism

- Various routes to structure

modification and function-

alization

- Lack of photostability

- Photobleaching

- Low molecular bright-

ness (i.e., low extinction

coefficient)

Designing and synthesizing molecular probes with

absorbance/emission in near-infrared (NIR) regions

for

- Endowing deeper penetration

- Improved spatial resolution

- Lower non-target autofluorescence

[47,103–

116]

Polymer

dots

- Biocompatibility

- Photostability

- Excellent brightness

- Fast radiative rate

- Large absorption coefficient

- Good water dispersibility

- Poor solubility/disper-

sion

- Low light penetration

depth

- Functionalizing the surface with appropriate chemi-

cal groups for enhanced solubility or further conju-

gation with other functional species

- Tuning the emission wavelength to situate in the

tissue transparency window

[8,14,49,117

–124]

Carbon-

based

quantum

dots

(QDs):

graphene

QDs and

carbon

dots

- Eco-friendly, facile synthe-

sis (carbon dots)

- Excellent photostability

- Biocompatibility

- Low cytotoxicity

- Low quantum yield

- Intrinsically hydropho-

bic surface

- Choosing appropriate precursors or post-synthetic

doping to gain desirable surface and photophysical

properties

- Energy-efficient method:

microwave and hydrothermal method

[8,21–

23,125–137]

Metal or-

ganic

frame-

work/co-

valent or-

ganic

frame-

works

- High porosity/large surface

- Tunable chemical structure

- High color purity

- Long lifetime

- Tunable chemical composi-

tion

- Possible modification post-

synthesis

- Available for diverse fields

- Difficult to control the

particle size and shape

for in vivo application

- Little information on bi-

ological activity

- Encapsulating guest species into the interior pore

- Post-synthetic modification to improve water solu-

bility and to control the photophysical properties

[138–154]

3. Small-Molecule Probes for Bioimaging

Small molecule-based organic fluorescent probes have a well-defined structure and

can be designed to be tunable for various applications according to their spectral proper-

ties based on their optical and physical characteristics. Because of these advantages, small

molecule-based organic fluorescent probes have been extensively studied for bioimaging.

There are two notable mechanisms that underly the luminescence processes at the molec-

ular level [155]. Mostly, small-molecule probes show concentration-dependent lumines-

cence behavior. Luminescence from the small molecules is weakened or quenched at high

concentrations. The quenching process can be associated with the formation of aggregates,

a phenomenon that has been referred to as aggregation-caused quenching (ACQ). Figure

3a presents a typical example of the ACQ effect. A diluted solution of N,N-dicyclohexyl-

1,7-dibromo-3,4,9,10-perylenetetracarboxylic diimide (DDPD) in tetrahydrofuran (THF)

is highly luminescent. However, its emission is weakened when water is added to THF.

Since ACQ is inconvenient for practical applications, numerous efforts have been devoted

to circumvent these limitations using chemical/physical methods and/or engineering pro-

cesses [156–160]. Currently, the design of new molecular systems with aggregation-in-

duced emission (AIE) is an active area of research on organic luminescent materials. Since

the AIE effect is opposite to the undesirable ACQ effect, it is important to actively utilize

Int. J. Mol. Sci. 2022, 23, 4949 8 of 52

the aggregation process instead of passively working against it. Figure 3b shows an ex-

ample of the AIE effect. When hexaphenylsilole is dissolved in a good solvent such as

THF, it is non-luminescent. However, intensive emission occurs after water is added to

THF. Recently, there have been many efforts to develop strategies for converting ACQ to

AIE. The Tang group reported that the restriction of intramolecular motions such as rota-

tions and vibrations is considered to be the main factor in overcoming the ACQ phenom-

enon [161]. Various AIE systems have been explored with decorated aromatic rings, flex-

ible chains, and intramolecular rings, such as coumarin, anthracene, naphthalene diimide,

pyrrole, pyridine, diketopyrrolopyrrole (DPP), porphyrin, and their derivatives [162–170].

Figure 3. Fluorescent digital photographs of (a) fluorescein and (c) hexaphenylsilole (HPS) solutions

with different bad solvent fractions. (b) Fluorescein molecules are disk-shaped and become non-

emissive when forming aggregates where a strong π−π interaction quenches the emission. (d) HPS

molecules are propeller-shaped and become highly emissive when forming aggregates where the

intramolecular rotation is restricted. Reprinted with permission from Ref. [155]. Copyright 2018,

American Chemical Society.

Likewise, understanding the role of the constituent chemical units in luminescent

probes is of vital importance. Table 2 lists representative key chemical units that constitute

the luminescent probes used for bioimaging. All these chemical units can play roles as

electron donors, acceptors, or bridges connecting the donor/acceptor. First, tetra-

phenylethylene (TPE), an AIE-active fluorescent probe, has been used in diverse studies

owing to its self-organization capability and ability to be incorporated into huge multi-

component assemblies with ACQ fluorescent probes. TPE can be functionalized with

guest organic species by electron conjugation. As a consequence, the absorption and

Int. J. Mol. Sci. 2022, 23, 4949 9 of 52

emission properties of the structures change, making them highly usable in various ap-

plications. For example, anthracene, pyrene, and carbazole have been combined with TPE,

and these functionalized forms of TPE exhibit distinct properties [171,172]. TPE has also

been combined with triphenylamine (TPA) to adjust the emission wavelength [173]. In

general, TPA is employed to achieve improved hole mobility. Benzobisthiadiazole (BBTD)

has been used to construct various NIR fluorescent probes owing to its excellent photo-

/thermostability and large Stokes shifts [174]. DPP has lactam rings that confer remarkable

light-harvesting properties [175]. In particular, DPP-based luminescent compounds or

materials can deliver outstanding photovoltaic performance in solar cells [176]. DPP with

an inherently low bandgap can be chemically modified or coupled with other materials

covalently or noncovalently (intermolecular π–π interaction), which offers possibilities to

tune luminescence characteristics (e.g., lowering the bandgap for absorbing and emitting

in the NIR region). Indocyanine green (ICG) is a well-known nontoxic NIR dye, exhibiting

absorption at 785 nm and emission at 800 nm. ICG was approved by the Food and Drug

Administration (FDA) in 1959 for evaluating hepatic function. Therefore, ICG is now

widely used clinically in medical diagnostics, photoacoustic imaging, photodynamic ther-

apy, and photothermal therapy. Thiophene consists of a five-membered heteroaromatic

compound containing a sulfur atom at one position. Although thiophene is insoluble in

water, it is soluble in many organic solvents. Because of the sulfur group, thiophene is

delocalized in the π-electron system. In addition, the abundant electrons of thiophene can

be used in various applications involving organic synthesis. Many theoretical and exper-

imental studies, including analyses of the inhibition efficiencies of thiophene and its de-

rivatives, have been performed to date. As a representative thiophene derivative, in-

deno[1,2-b]thiophene (IDT) is a powerful electron donor, which has a broad absorption

wavelength range as well as outstanding charge-transfer properties. This molecule not

only possesses an extended π-electron conjugation with excellent electron-donating capa-

bility due to its indole nitrogen atom but can further be synthesized with a one-step pro-

cess using commercially available materials. In addition, IDT contains a symmetry-break-

ing unit, which contributes to the photovoltaic performance of its derivatives. Another

thiophene derivative, 3,4-ethylenedioxythiophene (EDOT), acts as an electron donor and

is typically utilized after polymerizing with poly(3,4-ethylenedioxythiophene) (PEDOT).

Five-membered heterocycles, such as thiophene and pyrrole, and their derivatives have

been used as the building blocks to prepare conducting polymers [177–191]. Among the

existing conducting polymers, PEDOT shows particularly remarkable properties such as

high and stable conductivity, excellent transparency, and high water solubility (with pol-

ystyrene sulfonate). Therefore, PEDOT has been widely employed in various research

fields, ranging from a simple conductive coating to sensors, organic field-effect transistors,

organic light-emitting diodes, and photovoltaics [177–183,192]. Furthermore, many differ-

ent types of nitrobenzoxadiazole (NBD) fluorophore-based probes have been designed for

various sensing and biological applications, in which NBD is most frequently used to fa-

cilitate the ICT transition in the molecular system. The cyclopropenium cation is the clas-

sic example of a Hückel 2π-electron aromatic ring system, which is highly stable due to

its inherent aromaticity despite the apparent ring strain. Notably, the high degree of sta-

bility can be further enhanced through the incorporation of amino substituents onto the

cyclopropenium ring. Moreover, the photophysical properties of the inherently fluores-

cent cyclopropenium cations can be tuned with those of other π-conjugated groups (e.g.,

naphthalene) to increase the quantum yield, molar attenuation coefficient, Stokes shift,

and red-shifting absorbance and emission wavelengths. Lastly, triphenylphosphine

(TPP)-based luminescent compounds have rarely been reported to date due to the low

photoluminescence quantum yield. Recently, Zhang and colleagues [193] demonstrated

that introducing steric hindrance groups to the TPP moiety and separating the orbitals

involved in the transition can significantly suppress the non-radiative decay induced by

the structural distortion of TPP in the excited state and could further increase the quantum

yield by up to 0.89.

Int. J. Mol. Sci. 2022, 23, 4949 10 of 52

Table 2. Summary of the chemical units of small molecule-based photoluminescence probes a.

Full Name

(Abbreviation) Chemical Structure Characteristic Application Ref.

Tetraphenyleth-

ylene

(TPE)

- Capability of self-organization

- Conveniently incorporated into larger

multicomponent assemblies with fluoro-

phores

- OLEDs b

- Chemo-imaging

- Bioimaging

[194–197]

Benzobisthiadia-

zole

(BBTD)

- Strong electron acceptor

- Strong intramolecular charge transfer ef-

fect

- Sensitive to harsh synthetic procedures

- Organic field-effect transistors

- Photovoltaics

- OLEDs

[198–202]

Triphenylamine

(TPA)

- Good electron donor

- Carries a lone pair of electrons

- Electroluminescence

- Organic optoelectronics

- OLEDs

- Photovoltaics

[203–205]

Diketo-

pyrrolopyrrole

(DPP)

- Planar electron acceptor

- Good photostability/thermal stability

- High quantum yield

- Wide absorption spectrum

- High molar extinction coefficient

- Strong electron-withdrawing ability

- Large Stokes shift

- Easy modification

- Fluorescent sensing

- Photovoltaics

- Bioimaging

[206–211]

Indocyanine green

dye

(ICG)

- Nontoxic

- Near-infrared region of the absorp-

tion/fluorescence spectrum

- Wide fluorescence spectrum

- Reabsorption of itself due to overlap of

absorption/fluorescence

- Bioimaging

(e.g., determining cardiac out-

put, hepatic function, liver and

gastric blood flow, and for oph-

thalmic angiography)

[212–214]

Thiophene

- Numerous electrons


- A basic building block of organic semi-

conductor materials

- High photovoltaic performance

- Building block for organic

conductors

- Photovoltaics

[177,178,183

,215–218]

Indeno[1,2-b]thio-

phene

(IDT)

- Planar geometry

- Strong electron donation

- Can broaden the absorption wavelength

range as well as enhance the charge trans-

fer

- Provides a large and unoccupied π-sur-

face for effective intermolecular π−π inter-

actions.

- Photocatalyst

- OLEDs

- Organic field-effect transistors

- Photovoltaics

[219–221]

3,4-Ethylenedioxy-

thiophene

(EDOT)


- Mainly applied as a PEDOT c made

through polymerization

- Building block for organic

conductors

- Transparent conductive coat-

ing

- Electrode materials

- OLEDs

[177,178,183

,222]

Int. J. Mol. Sci. 2022, 23, 4949 11 of 52

- Photovoltaics

Nitrobenzoxadia-

zole

(NBD)

- Quite inexpensive

- Easily conjugated with large Stokes shift

- Good quantum yields

- Fluorescent donor

- Sensing applications

- Bioimaging

- Energy transfer

[199,223–

225]

Cyclopropenium

ion derivatives:

tris(dialkylamino)-

cyclopropenium

(TDAC) cation


- Outstanding stability

- Good charge delocalization

- Large Stokes shift

- Catalysts

- Super bases

- Ionic liquids

- Polyelectrolytes

- Photoelectronic redox-active

chemistries

[226–228]

Tri-

phenylphosphine

(TPP)

- Used as a phosphorus source

- Air-robust

- Cheap and good electron donor

- Catalyst for organic synthesis

- Electrocatalytic oxidation

- Water-splitting

[229–231]

a Note that the toxicity of a unit cannot be directly associated with that of fluorescent probes based

on the unit; b OLED: organic light-emitting diode; c PEDOT: poly(3,4-ethylenedioxythiophene).

Importantly, these unit structures described in Table 2 are employed as building

blocks to construct various molecular probes with ultraviolet (UV)-/NIR-range emissions

for different purposes and applications.

These core structures have rich π electrons in common. In one remarkable study per-

formed in 2017, Park et al. [232] utilized the π-abundant structure cyanine to detect hy-

drogen sulfide (Figure 4). Although hydrogen sulfide is a critical biological marker, few

studies have focused on its detection in vivo. Park et al. [232] investigated the difference

between absorption and emission peaks when their probe detected hydrogen sulfide. The

probe displayed three absorption peaks at 550–680 nm; however, when the probe detected

hydrogen sulfide, a new peak appeared at 700 nm. In the same vein, when the probe was

exposed to hydrogen sulfide, the fluorescence intensity increased sharply at 720 nm. They

further showed the highest occupied molecular orbital (HOMO), lowest unoccupied mo-

lecular orbital (LUMO) energy level, and bandgap; performed a selectivity test (Figure

4b); and obtained real-time fluorescence images in vivo using mice (Figure 4c).

Int. J. Mol. Sci. 2022, 23, 4949 12 of 52

Figure 4. (a) Chemical structure of a synthetic H2S-activated compound. (b) Fluorescence enhance-

ment of NIR-Az in the presence of thiol; reactive sulfur, nitrogen, and oxygen species; and ascorbic

acid. Reactions of (1) 10 μM NIR-Az with (2) 200 μM NaHS, (3) 1 mM L-cysteine, (4) 1 mM DL-

homocysteine, (5) 10 mM glutathione, (6) 200 μM Na2S2O3, (7) 200 μM Na2S2O4, (8) 200 μM Na2SO3,

(9) 200 μM Na2SO4, (10) 200 μM NaHSO3, (11) 200 μM KSCN, (12) 200 μM H2O2, (13) 200 μM Angeli’s

salt (NO−), (14) 200 μM NaNO3, (15) 200 μM NaNO2, (16) 200 μM ascorbic acid, and (17) 200 μM α-

lipoic acid in PBS buffer (10 mM, pH 7.4, 30% acetonitrile, v/v) at 37 °C for 30 min (λex = 680 nm,

λem = 720 nm). (c) Fluorescence images of 10 μM NIR-Az in the presence of thiol; reactive sulfur,

nitrogen, and oxygen species; and ascorbic acid (λex = 675 nm, λem = 700 nm). (d) Representative

fluorescence images of mice given i.p. injections of the NIR-Az probe (50 μM, in 20 μL of DMSO)

and then injected with different amounts of NaHS: (i) 0, (ii) 5, (iii) 10, and (iv) 20 equivalents of

NaHS (in 100 μL of PBS). (e) Quantification of the fluorescence emission intensity from the ab-

dominal area of the mice of groups i–iv in (d). (f) Representative fluorescence images of mice ad-

ministered an i.p. injection of the probe NIR-Az (50 μM, in 20 μL of DMSO) and then injected with

10 equivalents of NaHS (100 μM, in 100 μL of PBS). Images were taken after incubation of NaHS for

(i) 0, (ii) 0.5, (iii) 1, (iv) 2, and (v) 3 h. (g) Quantification of the fluorescence emission intensity from

the abdominal area of the mice of groups i–v in (f). Reprinted with permission from Ref. [232]. Copyright 2017, Elsevier.

3.1. UV Small-Molecule Probes

As an excitation energy, short-wavelength UV photons may induce bond cleavage,

isomerization, or rearrangement reactions in organic and even inorganic molecules.

Therefore, UV excitation presents several challenges, particularly in biological applica-

tions. For example, high-energy UV light has highly limited tissue penetration due to high

Int. J. Mol. Sci. 2022, 23, 4949 13 of 52

optical scattering and absorption by endogenous chromophores, which can lead to sample

overheating and in turn cause phototoxic or photoallergic reactions.

Examples of UV probes developed in recent studies are presented in Figure 5, which

were mainly designed for the purposes of bioimaging DNA and HeLa cells in an in vitro

context. This is considered to be due to the lower in vivo permeability of UV probes than

NIR probes. Probes 1 to 11 consist of a cyclopropenium core and three primary amine

arms, varying in size, conjugation, and structural flexibility. Remarkably, probes 10 and

11 have large Stokes shifts of ~180 nm, whereas most UV probes have small Stokes shifts

(probes 1 to 9: <l00 nm). Therefore, the use of probes 10 and 11 can achieve enhanced

sensitivity by avoiding self-reabsorption effects. As illustrated in Figure 6, probes 10 and

11 have larger Stokes shifts due to the combination of two excitation processes: (i) ICT

from the cyclopropenium core (electron donor) to the appended arms (electron acceptor)

and (ii) intramolecular through-space conjugation among the appended arms. Neverthe-

less, it is important to note that molecular fluorophores suffer from ACQ when they are

aggregated. To circumvent this effect, Montalti et al. [103] modified NBD (see Table 2)

with triphenyphosphazene groups (probes 12, 13, and 14). Probes 13 and 14 successfully

self-organized as highly bright and fluorescent nanoparticles with high colloidal stability

in phosphate-buffered saline solution. Notably, in the aqueous phase, probe 13 nanopar-

ticles displayed a brightness that was more than six orders of magnitude higher than that

of the molecular counterpart in the organic solvent.

Figure 5. Chemical structure of organic probes (probes 1 to 14) for bioimaging in the ultraviolet range.

Int. J. Mol. Sci. 2022, 23, 4949 14 of 52

Figure 6. Schematic representation of intramolecular charge transfer (ICT) and intramolecular through-

space conjugation (ITSC) and the coupling of these processes in trisaminocyclopropenium fluorophores.

Reprinted with permission from Ref. [105]. Copyright 2020, American Chemical Society.

These fluorescent probes belonging to the UV range have mainly been studied in the

context of in vitro research and to determine their photophysical characteristics. First, Fig-

ure 7a–g shows a recent, notable example of the application of probe 10 for biolabeling

[105]. The researchers hypothesized that this hydrophobic tris-amino cyclopropenium

small-molecule phosphor could function as a membrane counterstain in mammalian cells.

To test this hypothesis, probe 10, which was dissolved in dimethyl sulfate oxide (DMSO),

was added to HEK293 (human embryonic kidney) cells at concentrations of 10 nM, 100

nM, and 10 μM. Surprisingly, probe 10 formed membrane-related spheres outside the cell

under all concentrations tested (Figure 7a(i)). Although the properties of these spheres

were not examined in detail, it was assumed that the solution contains probe 10 based on

fluorescence emission profiles (Figure 7a(ii),b(ii),c(ii)), consistent with the effects of probe

10. Spheres containing probe 10 formed as a result of phase separation between the hy-

drophobic probe 10 and water-soluble culture medium, and they were associated with

hydrophobic cell membrane lipid bilayers through hydrophobic interactions. In addition

to the fluorescence emitted from some membrane-related spheroid cells, minimal fluores-

cence release was observed in cell membranes treated with 10 or 100 nM probe 10 solu-

tions after excitation at 380 nm (Figure 7b(i),c(i)). However, cells treated with 10 μM of

probe 10 showed bright fluorescence emissions at 450 nm in what appeared to be nuclei

(Figure 7b(i),c(i)) and showed no morphological differences from those of controls before

cell medium removal (Figure 7a–g). To verify this finding, HeLa cells (Henrietta Lacks,

cervical cancer cell line) were implanted with DNA plasmids containing genetically en-

coded expression structures capable of inducing the cellular production of the red fluo-

rescent protein mScarlet carrying orthogonal fluorescence sequences (mScarlet-NLS).

Conversely, upon expression, mScarlet-NLS showed a distinct fluorescence excita-

tion/emission profile compared with that of probe 10, which can be used to visually de-

compose the two. After treating the HeLa cells expressing mScarlet-NLS with probe 10,

450 nm fluorescence was clearly visualized as mScarlet fluorescence and co-localized in

the cell nucleus (Figure 7d–g). In contrast, cells treated only with DMSO exhibited mScar-

let fluorescence but did not exhibit fluorescence at 450 nm. Interestingly, in contrast to the

hypothesis that probe 10 would function as a fluorescent membrane counterstain, specific

labeling and localization of probe 10 to the cell nucleus were observed. This study adds to

the burgeoning interest in cyclopropenium compounds and their use as fluorophores in

bioimaging applications.

Int. J. Mol. Sci. 2022, 23, 4949 15 of 52

Figure 7. (a–c) Probe 10 fluorescently labels the nuclei of cells. HEK293 cells were imaged 5 min

post-treatment in (a-i, b-i, c-i) 1% DMSO cell media, (a-ii, b-ii, c-ii) 1% DMSO and 10 nM probe 10

cell media, (a-iii, b-iii, c-iii) 1% DMSO and 100 nM probe 10 cell media, or (a-iv, b-iv, c-iv) 1% DMSO

and 10 μM probe 10 cell media. (a-i, ii, iii, iv) Bright-field images. (b-i, ii, iii, iv) A 380 nm excitation

channel and 450 nm emission fluorescence channel to image probe 10. (c-i, ii, iii, iv) Merged images

of bright-field and fluorescent images. Spherules (white arrows) are seen to form on the cell mem-

branes at all concentrations tested of probe 10 (10 nM, 100 nM, and 10 μM) but fail to be observed

when only DMSO was present. Several of these spherules produce significant amounts of blue flu-

orescence (yellow arrows), indicating the presence of probe 10. The nuclei (green arrows) can be

visualized following 10 μM treatment with probe 10. (d–g) Validation of probe 10 for nuclear stain-

ing. HeLA cells were transfected with an mScarlet-NLS expression construct and imaged 5 min post-

treatment in (d-i, e-i, f-i, g-i) 1% DMSO cell media or (d-ii, e-ii, f-ii, g-ii) 1% DMSO and 10 μM probe

10 media. (d-i, d-ii) Bright-field image. (e-i, e-ii) A 380 nm excitation channel and 450 nm emission

fluorescence channel to image probe 10. (f-i, f-ii) A 570 nm excitation channel and 600 nm emission

fluorescence channel to image mScarlet-NLS. (g-i, g-ii) Merged images of all channels in the treat-

ment condition. The nuclei (green arrows) can be visualized following 10 μM treatment, which over-

laps with fluorescence being emitted from nuclei indicated by mScarlet-NLS (magenta arrows). Re-

printed with permission from Ref. [105]. Copyright 2020, American Chemical Society. (h) Fluores-

cence signal of probe 13 and 14 nanoparticles in cells. HeLa cells grown on glass coverslips were

incubated for 20 h with no nanoparticles (medium alone), probe 13 nanoparticles, or probe 14 nano-

particles and analyzed by fluorescence confocal microscopy with the same instrumental setting for

comparison. Representative images are shown. In the case of probe 13 nanoparticles, cell signal dis-

tribution details are shown at higher magnification. Reprinted with permission from Ref. [103]. Copyright 2019, Frontiers Media SA.

Figure 7h exhibits a representative application example of probe 13 and 14 nanopar-

ticles. Probe 13 and 14 nanoparticles were cultured with HeLa cells at a relatively low dose

of 80 ng mL−1 at 37 °C to prove their effectiveness as fluorescent probes. After 20 h, confo-

cal microscopy analysis showed that probe 13 nanoparticles provided an intense, struc-

tured signal within the cell cytoplasm, indicating endosomal internalization of the

Int. J. Mol. Sci. 2022, 23, 4949 16 of 52

nanoparticles. Compared with those of probe 13 nanoparticles, probe 14 nanoparticles

showed much weaker signals due to their intrinsically weak fluorescence characteristics.

In addition, these probes showed that both probe 13 and 14 nanoparticles had no toxic

effects at up to 1 g mL−1 in HeLa cells through separate viability assays, involving use of

a colorimetric reagent (MTS) for the quantification of viable cells. In other words, the cel-

lular experiments proved that probe 13 and 14 nanoparticles are suitable as fluorescent

contrast agents for bioimaging owing to their good biocompatibility. These results further

demonstrated that the rational design of a molecular precursor is fundamental for pro-

ducing stable and intensely bright aggregate nanoparticles.

3.2. NIR Small-Molecule Probes

Compared to UV light, NIR light can penetrate deeper into tissues without inducing

any photochemical damage. In addition, NIR light sources are usually cheaper, more com-

mon, and more accessible to non-specialist end users. Figure 8 exhibits molecular probes

for which their fluorescence emissions belong to the NIR region, which has been reported

in recent studies [47,111,112,116]. As described above, probes belonging to the NIR region

are more suitable for in vivo applications because they more easily penetrate cells than

probes belonging to the UV region. Although the concept of small molecule-based NIR

fluorophores is simple, their synthesis and purification require multiple steps and mostly

result in low yields. Therefore, optimizing the procedures for the design and synthesis of

the molecular probes remains a challenge for organic chemists.

Figure 8. Scheme of chemical structures (probes 15–23) for bioimaging in the near-infrared range.

The major strategies for designing NIR molecular probes can be summarized as fol-

lows. NIR-I probes have been mostly obtained with a donor–acceptor (D–A) system. The

combination of strong electron donors and acceptors in a molecule may lower the

bandgap energy, resulting in NIR-I probes with emission wavelengths of ~900 nm.

Int. J. Mol. Sci. 2022, 23, 4949 17 of 52

Moreover, further introducing a second strong donor in the D–A architecture allows

yielding a symmetrical donor–acceptor–donor (D–A–D) architecture. The strong electron-

donating groups adjacent to the central electron acceptor reduce the energy gap between

hybridized HOMO/LUMO levels, which drives the fluorescence emission into the NIR-II

region (emission wavelengths longer than 1000 nm). Representatively, BBTD, which is a

strong electron-deficient unit, has been widely used as an acceptor. However, owing to

the strong ICT effect, BBTD-based fluorophores suffer from low quantum yields in aque-

ous solution. Therefore, there is a demand for the development of new acceptor units to

afford more alternative design strategies of NIR-II molecular probes. The combination of

various spacers and electron donors/acceptors can provide unique opportunities to ex-

pand the library of small molecule-based NIR-II fluorophores. Table 3 summarizes the

absorption peak, fluorescence emission peak, and Stokes shifts of the small-molecule

probes that have been recently reported in bioimaging studies. Most of these studies have

calculated the HOMO/LUMO levels and the resulting bandgap energies through density

functional theory, which are also given in Table 3.

Table 3. Summary of the optical properties with small molecule-based photoluminescence probes.

Material λex

(nm)

λem

(nm)

Stokes Shifts

(nm)

HOMO a

(eV)

LUMO b

(eV) Band Gap (eV) Ref.

1 339 422 83 –0.27 –0.10 0.17

[105]

2 242 307 65 –0.30 –0.13 0.17

3 274 304 30 –0.30 –0.17 0.12

4 250 335 85 -- -- --

5 249 338 89 –0.28 –0.17 0.11

6 278 306 28 –0.28 –0.17 0.11

7 274 306 32 –0.28 –0.17 0.11

8 250 340 90 –0.28 –0.17 0.11

9 337 421 84 –0.28 –0.20 0.08

10 248 428 180 –0.28 –0.20 0.08

11 248 431 183 –0.28 –0.20 0.08

12 482 536 54 -- -- --

[103] 13 488 526 38 -- -- --

14 524 555 31 -- -- --

15 730

730 (NPs c)

--

898 (NPs)

--

168 (NPs) -- -- --

[111]

16 808

730 (NPs)

--

--

--

-- -- -- --

17 610 665 55 -- -- --

[116] 18 700 900 200 -- -- 1.91

19 700 900 200 -- -- 1.82

20 643 922 279 –5.45 –3.67 1.78

[47] 21 762 1062 300 –4.84 –3.39 1.45

22 725 1050 325 –4.83 –3.35 1.48

23 805 1034 229 –4.50 –3.35 1.15 [112] a HOMO: highest occupied molecular orbital; b LUMO: lowest unoccupied molecular orbital; c NPs:

nanoparticles.

3.3. Nanoparticulate Small-Molecule Probes

To improve in vivo bioavailability, nanoparticulate molecular fluorophores can be

prepared by physically encapsulating or chemically conjugating/modifying them with bi-

ocompatible polymers and peptides. Figure 9 provides examples of preparing nanopar-

ticulate small molecule-based probes from several studies.

Int. J. Mol. Sci. 2022, 23, 4949 18 of 52

Figure 9. (a-i) Size distribution obtained by dynamic light scattering (DLS) analysis of nanoparticles

from probe 13. (a-ii) Representative transmission electron microscopy (TEM) image of probe 13 na-

noparticles. (a-iii) Size distribution resulting from the analysis of the TEM images of probe 13 nano-

particles. (a-iv) Size distribution obtained by DLS analysis of probe 14 nanoparticles. (a-v) Repre-

sentative TEM image of probe 14 nanoparticles. (a-vi) Size distribution resulting from the analysis

of TEM images of probe 14 nanoparticles. Reprinted with permission from Ref. [103]. Copyright

2019, Frontiers Media SA. (b–i) Schematic illustration of probe 15 nanoparticles. (b-ii) TEM images

of probe 15 nanoparticles. (b-iii) Size measurement of probe 15 nanoparticles in water using DLS.

Reprinted with permission from Ref. [111]. Copyright 2021, Royal Society of Chemistry. (c-i) Sche-

matic illustration of probe 19 encapsulating nanoparticles by Pluronic F127. The DLS spectrum of

(c-ii) probe 17 nanoparticles with F127, (c-iii) probe 18 nanoparticles with F127, and (c-iv) probe 19

nanoparticles with F127 (inset: TEM image of nanoparticles). Reprinted with permission from Ref.

[116]. Copyright 2021, John Wiley and Sons. (d-i) Formation of probe 22 nanoparticles via nanopre-

cipitation. (d-ii) DLS and TEM images of probe 22 nanoparticles; scale bar: 100 nm. Reprinted with

permission from Ref. [47]. Copyright 2021, Royal Society of Chemistry. (e-i) Schematic illustration

of the preparation method of NIR-II AIE nanoparticles (probe 23 nanoparticles) via micellization.

(e-ii) Representative TEM image of probe 23 nanoparticles. Scale bar: 100 nm. Reprinted with per-

mission from Ref. [112]. Copyright 2021, Royal Society of Chemistry.

Int. J. Mol. Sci. 2022, 23, 4949 19 of 52

First, Figure 9a exhibits the morphology of nanoparticles prepared from probes 13

and 14 using a nanoprecipitation method [103]. Transmission electron microscopy (TEM)

images showed that the probe 13 and 14 nanoparticles have diameters of 91 ± 13 and 54 ±

9 nm, respectively. The diameter measured for probe 13 nanoparticles by TEM was con-

sistent with the hydrodynamic diameter measured by dynamic light scattering (DLS).

However, a large difference between the TEM and DLS diameters was observed in the

case of probe 14 nanoparticles, indicating partial inter-nanoparticle aggregation in aque-

ous solution. The hydrodynamic diameter is much more important in practical applica-

tions because it more precisely reflects how the nanoparticles move in a fluid. Next, using

probes 15 and 16, water-dispersible nanoparticles were also fabricated using a modified

nanoprecipitation method (Figure 9b) [111]. TEM images indicated that these nanoparti-

cles are spherical in shape with a narrow particle size distribution. DLS analysis revealed

that the nanoparticles with hydrodynamic diameters of 52.5 ± 3.4 and 55.7 ± 4.2 nm were

prepared from probes 15 and 16, respectively. To improve biocompatibility, probes 17, 18,

and 19 were encapsulated into nanoparticles by a pluronic surfactant (F127) through na-

noprecipitation (Figure 9c) [116]. The pluronics correspond to polyethylene oxide-poly-

propylene oxide-polyethylene oxide-based triblock copolymers, which are available to

serve as non-ionic surfactants with varying compositions and hydrophilic–lipophilic bal-

ance. In particular, Pluronic F127 is the most popular pluronic for various biological ap-

plications because it is approved by the FDA as an excipient in oral, ophthalmic, and top-

ical medicinal formulations. TEM revealed that the morphologies of the nanoparticles fab-

ricated from probes 17, 18, and 19 were spherical. The DLS diameters of the nanoparticles

were found to be approximately 30 nm. During storage at 4 °C for 20 days, no significant

variations of the zeta potential and polymer dispersity index were observed, indicating

the excellent stability of these nanoparticles.

In probes 20−22, the substituted hexyloxy chains serve as a strong donor, and other

electron-withdrawing or electron-donating groups such as nitrobenzene (probe 20), ami-

nobenzene (probe 21), and triphenylamine (probe 22) were also introduced as substituted

groups into the probes [47]. Importantly, in probe 22, 3,4-bis(hexyloxy)thiophene and tri-

phenylamine serve as the first donor and second donor, respectively, for AIE. Probe 21

and 22 nanoparticles with high monodispersity and homogeneity were also prepared us-

ing a nanoprecipitation method with the aid of a DPPE-5KPEG amphiphile (Figure 9d).

Representatively, probe 22 nanoparticles were characterized for bioimaging applications

using TEM and DLS, showing an average size of ~90 nm and a dynamic size of ~120 nm.

Probe 23, based on BBTD and TPE, shows NIR-II fluorescence with AIE characteristics

[112]. To illustrate the feasibility of probe 23 for bioimaging application, similarly to

probes 21 and 22, water-soluble and biocompatible nanoparticles were fabricated with a

nanoprecipitation method using an amphiphile (DSPE-PEG5000) (Figure 9e). Briefly, a

mixture of probe 23 and THF was rapidly added into an aqueous DSPE-PEG5000 solution

in an ice bath under continuous sonication; then, the remaining THF and DSPE-PEG5000

were carefully removed. The resulting nanoparticles exhibited high monodispersity and

homogeneity with an average particle size of ~50 nm, as measured by TEM, and a hydro-

dynamic diameter of ~60 nm, as measured by DLS.

3.4. In Vivo Bioimaging

To date, many strategies for in vivo bioimaging using small molecule-based probes

have been developed to obtain information about biological functions and diseases [233].

As an example, Xu and co-workers [234] reported a two-photon-triggered NIR fluores-

cence technique for cancer theranostics (imaging and photodynamic/gene therapy) using

small-molecule probes with large π-conjugated TPA derivatives and a lipophilic tail (Fig-

ure 10). Notably, the unique molecular architecture allowed TPA-based probes to acquire

NIR AIE characteristics, large Strokes shifts, and intense two-photon excitation fluores-

cence. In addition, the TPA-based probe molecules assembled together with DNA to form

nanoparticles, triggering the AIE property. Consequently, TPA-based probes showed

Int. J. Mol. Sci. 2022, 23, 4949 20 of 52

high two-photon excitation on four different types of cell lines in the absence/presence of

serum and were capable of tracking the transfection process and monitoring the real-time

in vivo biodistribution with high resolution and deep penetration. In vivo bioimaging us-

ing the TPA-based probe with DNA to tumor-bearing nude mice displayed bright fluo-

rescence over time, indicating that the probe has excellent tumor retention properties as

well as long-term biocompatibility, demonstrating the promise of two-proton fluores-

cence using the TPA-based probe for bioimaging and theranostics.

Figure 10. (a) Structures of probe molecules consisting of three parts: two polar [12]aneN3-triazole

heads (blue), a luminogen core (red), and a long hydrophobic tail (black). (b) Ex vivo one-photon or

two-photon images of the tumor tissue injected with [12]aneN3-modified TPA derivatives/pUC18

DNA. (c) In vivo imaging at different time points after intratumor injection with [12]aneN3-modified

TPA derivatives/pUC18 DNA. (d) Ex vivo biodistribution of various organs and tumor tissue from

mice 24 h post-injection with [12]aneN3-modified TPA derivatives/pUC18 DNA. (e) Major organs

harvested from the mice after being intratumorally injected with [12]aneN3-modified TPA deriva-

tives/pUC18 DNA for 21 days. Reprinted with permission from Ref. [234]. Copyright 2021, Ameri-

can Chemical Society.

There are also several remarkable studies on in vivo bioimaging using small mole-

cules, as described above. Figure 11 summarizes the results of a recent study that investi-

gated NIR-II fluorescence imaging using small-molecule probe 15. To evaluate the poten-

tial of probe 15 nanoparticles for imaging of the animal circulatory system, Li and col-

leagues [111] performed vascular imaging in mice through both NIR-II fluorescence and

NIR-I photoacoustics using an animal fluorescence imaging system and photoacoustic mi-

croscopy (PAM), respectively. Real-time video-rate NIR-II fluorescence imaging of the

vascular networks in the ears, hindlimbs, and brain was performed on C57BL/6J mice fol-

lowing intravenous injection of probe 15 nanoparticles (1000 LP). At 10 min after admin-

istration of the nanoparticles (50 mg per mouse), the vasculature from the hindlimbs, ears,

and brain could be clearly visualized with high resolution under 785 nm excitation at a

power density of 100 mW cm−2. The cross-sectional fluorescence profiles of the representa-

tive vessels were plotted with the physical location along the white lines. The results

Int. J. Mol. Sci. 2022, 23, 4949 21 of 52

suggested that the fluorescence profiles from the blood vessels of the hindlimbs, ears, and

brain have a full width at half maximum (FWHM) of 76.0, 71.6, and 110.3 μm, respectively,

indicating the high sensitivity and spatial resolution. Cerebral vessel imaging was also

performed under an intact skull. Upon subcutaneous injection of probe 15 nanoparticles

through the flank of a mouse, the adjacent lymph node to the injection site was lit up,

providing a high-resolution image of the lymphatic vessel with an FWHM of 454.0 μm

(Figure 11e–g). PAM was employed to further demonstrate the high spatial and temporal

resolution for in vivo vasculature imaging provided by probe 15 nanoparticles. Nude mice

were anesthetized and placed on the imaging stage before imaging blood vessels in the

ear and brain. Upon intravenous administration, the blood vessels were lit up and clearly

observed upon 730 nm pulse laser excitation (Figure 11h–i). PAM indicated that the

FWHM of the ear blood vessels was 223.0 μm. In brain vessel imaging upon scalp removal,

both the transverse sinus and superior sagittal sinus were clearly visualized (Figure 11i).

In addition, the inferior cerebral veins also emitted photoacoustic signals upon probe 15

nanoparticle injection, whereas they were nearly undetectable before probe 15 nanoparti-

cle injection. The tumor-targeting ability of the nanoparticles was then examined in nude

mouse models subcutaneously inoculated with 143B osteosarcoma cells and PC3 prostate

carcinoma cells (Figure 11j–l). For each type of tumor, two groups of tumor-bearing mice

were used to confirm the targeting ability of probe 15 nanoparticles comprising Arg–Gly–

Asp peptide (RGD) (n = 4 per group). The first group of mice was injected with probe 15–

RGD nanoparticles, while another control group of tumor-bearing mice was treated with

probe 15 nanoparticles. The NIR-II fluorescence signals from the tumor sites gradually

increased and reached a maximum at 48 h post injection of probe 15–RGD nanoparticles

in both tumor-bearing mouse groups (Figure 11j,k). However, the signals from tumor sites

in mice injected with probe 15 nanoparticles revealed only limited enhancement within

the test period. Additionally, the fluorescence intensity from the tumor sites of the mice

injected with probe 15–RGD nanoparticles was higher than that from the control group at

all time points. Quantification suggested that the maximum average signal-to-back-

ground (T/NT) ratios of probe 15–RGD nanoparticle-treated mice were ~14.2 and ~8.1 in

the 143B and PC3 tumor models, respectively, while those of the corresponding probe 15

nanoparticle-treated mice were only 5.0 and 4.9 at 72 h post injection. The 143B tumor-

bearing mice were sacrificed to collect the vital organs and tissues for imaging. The quan-

titative analysis of ex vivo fluorescence imaging results suggested that all these organs

and tissues from mice injected with probe 15–RGD nanoparticles emitted fluorescence

with different signal intensities. In particular, the injected probe 15–RGD nanoparticles

were mainly distributed in the tumor, liver, spleen, and lymph node. The skin sample also

showed fluorescence signals, indicating the retention of probe 15–RGD nanoparticles due

to high binding affinity with the integrin AVB3 expressed in the skin tissues. In addition

to fluorescence imaging, photoacoustic tomography was also performed to further con-

firm the overall targeting effect of probe 15–RGD nanoparticles at tumor sites (Figure 11l).

The photoacoustic signal intensity at the tumor site from a probe 15–RGD nanoparticle-

treated mouse was also higher than that of a probe 15 nanoparticle-treated mouse, which

was consistent with the results obtained from NIR-II fluorescence imaging. These results

successfully demonstrated the high potential of probe 15 nanoparticles in dual-modality

bioimaging applications. Thus, by taking advantage of the high sensitivity of NIR-II fluo-

rescence imaging, a clear visualization of tumor margins in a two-dimensional view was

successfully achieved.

Int. J. Mol. Sci. 2022, 23, 4949 22 of 52

Figure 11. Probe 15 nanoparticle-based imaging in vivo. (a) Schematic illustration of intravenous

injection of the probe nanoparticles through the tail vein of a mouse. (b–d) Representative NIR-II

fluorescence images of the hindlimb vasculature (b), ear vessels (c), and brain vessels (d) in the

mouse upon intravenous injection of 100 mg of the probe nanoparticles. (e) Schematic illustration

of subcutaneous injection of the probe nanoparticles for lymphatic imaging. (f) Representative NIR-

II fluorescence image of the lymphatic vessels and lymph nodes in a mouse injected with the nano-

particles. (g) Cross-sectional fluorescence intensity profile (and Gaussian fit) along the red line, cir-

cled in green, in panel (f). PAM imaging of the ear vessels (h) and brain vessels (i) from a mouse

injected with the probe nanoparticles through the tail vein. (j) Representative NIR-II FLI in 143B

tumor-bearing mice using the probe-based nanoparticles over 72 h under excitation by an 808 nm

diode laser (140 mW cm−2); filter: 1000 nm LP. Arrows indicate the subcutaneous tumor (n = 4 per

group). Scale bar: 5 mm. (k) T/NT ratios in probe nanoparticle-based 143B tumor imaging over 72

h. Data are plotted as mean and standard deviation (n = 4). (l) Three-dimensional volume rendering

of photoacoustic images (excitation at 730 nm) of the 143B tumor in a mouse 48 h post-injection of

the probe nanoparticles without (left) or with (right) RGD peptide decorated on the surface. Scale

bar = 2 mm. Reprinted with permission from Ref. [111]. Copyright 2021, Royal Society of Chemistry.

Next, as shown in Figure 12a, photoluminescence angiography was successfully re-

alized as a non-radioactive imaging strategy by intravenously injecting probe 19 nanopar-

ticles with Pluronic F127 [116]. NIR-II (900–1700 nm) and NIR-IIa (1300–1400 nm) fluores-

cence images of whole-body blood vessels in mice were acquired with different filters

(900–1300 nm) after intravenous injection of the probe nanoparticles (200 μL, 0.5 mg mL−1).

With an increase in the wavelengths of the long-pass (LP) filter, the spatial resolution of

images considerably improved. Higher-resolution angiography images of the mouse were

obtained in the NIR-IIa region with a Gaussian-fitted FWHM of 0.50 mm and SBR of 1.77.

The distance between the camera and camera lens was increased to acquire magnified

images. NIR-IIa fluorescence imaging was then further applied to the hindlimb blood ves-

sels. As displayed in Figure 12i, the NIR-II fluorescent image (1100 nm LP filter) displayed

the clear morphology of blood vessels. To evaluate the quality of NIR-II fluorescence im-

aging, a line was drawn across the femoral vessels, and Gaussian-fitted analysis was per-

formed. The FWHM of the femoral vessels was 0.60 mm, and SBR was 1.90. The femoral

artery and femoral vein, which are very close and thus difficult to distinguish with the

1100 nm LP filter, were clearly observed in the NIR-IIa region (1300 nm LP filter) with

Int. J. Mol. Sci. 2022, 23, 4949 23 of 52

higher resolution (femoral artery, FWHM1 = 0.29 mm; femoral vein, FWHM2 = 0.51 mm;

SBR = 2.88). In addition, cerebral blood vessel imaging was further carried out in mice

after performing a craniotomy. As presented in Figure 12k, due to the high SBR of bioim-

aging in the NIR-II region (1100 nm LP), the cerebral blood vessels were clearly imaged

by an InGaAs detector (FWHM = 100 μm, SBR = 2.47) after the intravenous injection of the

probe nanoparticles (200 μL, 500 μg mL−1). In NIR-IIa fluorescence images (1300 nm LP)

of the cerebral blood vessels, the same cerebrovasculature was observed at higher spatial

resolution (FWHM = 87 μm, SBR = 3.93), which further confirmed that fluorescence imag-

ing in the NIR-IIa region can render better clarity and resolution compared with that in

the NIR-II region.

Figure 12. Probe 19 nanoparticle-based imaging in vivo. (a) Schematic illustration of NIR-II fluores-

cence imaging of the hindlimb vasculatures in BALB/c nude mice using a wide-field imaging sys-

tem. (b) Comparison of NIR-II fluorescence signals at different concentrations with different LP fil-

ters (0.1, 0.25, and 0.5 mg mL−1). (c–g) NIR-II images of the whole-body vessels of mice after intra-

venous injection of the probe 19 nanoparticles with F127 under irradiation with a 690 nm laser (200

μL, 0.5 mg mL−1). Scale bar = 2 cm. (h) Photographs of the devices and instruments used in hindlimb

Int. J. Mol. Sci. 2022, 23, 4949 24 of 52

and brain blood vessel imaging experiments. (i) NIR-II and (j) NIR-IIa fluorescence images of the

hindlimb blood vessels of nude mice using 1100 nm and 1300 nm LP filters, respectively. Scale bar

= 5 mm. (k) NIR-II and (l) NIR-IIa fluorescence images of the brain blood vessels during craniotomy

in C57BL/6 mice using 1100 nm and 1300 nm LP filters, respectively. (Scale bar = 3 mm). Reprinted

with permission from Ref. [116]. Copyright 2021, John Wiley and Sons.

NIR-II fluorescent probe 23 nanoparticles with the AIE property were also available for

visualizing tumor-feeding blood vessels, long-term hindlimb vasculature, and incomplete

hindlimb ischemia, as well as long-term breast tumor imaging. Figure 13 summarizes the

data of in vivo NIR-II fluorescence imaging for breast tumors and tumor-feeding blood ves-

sels using probe 23 nanoparticles [112]. The in vitro cellular endocytosis of probe 23 nano-

particles was examined using 4T1 breast cancer cells. The NIR-II fluorescence intensity of

the 4T1 cells incubated with probe 23 nanoparticles revealed explicit time dependence, in-

dicating that probe nanoparticles can permeate cells via the cellular membrane. To examine

the ability of the probe nanoparticles to diagnose breast cancer early, a mouse model of

breast cancer was established in which 4T1 breast cancer cells (approximately 2 × 106) were

subcutaneously injected into the right leg of female BALB/c mice. NIR-II fluorescence imag-

ing of 4T1 breast tumors was conducted by intravenously injecting the probe nanoparticles

(0.2 mL, 10 mg kg−1) into 4T1 tumor-bearing mice (n = 3) through the tail vein when the

tumors reached ~60 mm3 in 1 week. The fluorescence intensity (1000 nm LP, 100 ms) in tu-

mors reached the highest level at 48 h post-injection under 808 nm excitation with a power

density of 90 mW cm−2 (Figure 13d). For up to 8 days post-injection, the NIR-II fluorescence

signal of tumors was very strong and clearly delineated the normal tissues, demonstrating

the suitability of the probe nanoparticles for long-term tumor imaging and real-time image-

based monitoring in solid tumor therapy. An ex vivo biodistribution study was also carried

out to evaluate the distribution of the probe nanoparticles in the major organs. High accu-

mulation was observed in the liver and spleen, indicating that the probe nanoparticles

would be cleared through the hepatobiliary system.

The probe 23 nanoparticles were then intravenously injected into 4T1 tumor-bearing

BALB/c mice (n = 3) to evaluate their feasibility as an NIR-II contrast agent for the in vivo

imaging of the tumor-feeding blood vessels. At 2 min post-injection, the tumor vascula-

ture was successfully visualized using an InGaAs camera with 1000 nm (Figure 13b, 200

ms exposure time) and 1250 nm (Figure 13c, 800 ms exposure time) LP filters under 808

nm laser excitation. NIR-II fluorescence images of the 4T1 tumor blood vessels showed a

more distinct vascular network with the 1250 nm LP filter than with the 1000 nm filter,

due to both reduced biological auto-fluorescence and photon scattering in the longer-

wavelength NIR-II region. Moreover, a similar image quality via different LP filters was

demonstrated in the hindlimb vasculature of C57BL/6 mice (Figure 13d,e). Surgery was

conducted to induce incomplete hindlimb ischemia to mimic the NIR-II fluorescence im-

aging-guided vascular embolization procedure, and the occlusion site of blood supply

vessels was precisely monitored by NIR-II imaging using the probe nanoparticles (Figure

13f). The capability of long-term vasculature imaging using the probe nanoparticles was

then examined. As exhibited in Figure 13g, the vasculature was still clearly visualized

through NIR-II imaging at 4 h post-injection. Furthermore, the resolution (497.6 mm) of

the hindlimb vasculature calculated through the Gaussian-fitted FWHM confirmed the

capability of the probe nanoparticles to precisely map vascular vessels with NIR-II imag-

ing (Figure 13h).

Int. J. Mol. Sci. 2022, 23, 4949 25 of 52

Figure 13. Probe 23 nanoparticle-based imaging in vivo. (a) Representative in vivo long-term NIR-

II fluorescence images (808 nm excitation, 90 mW cm−2; 1000 nm LP, 100 ms; n = 3) of the 4T1 breast

tumor model at different time points after tail vein injection of the probe nanoparticles (0.2 mL, 10

mg kg−1). The white circles indicate the location of the 4T1 tumor. Scale bar: 1 cm. (b,c) In vivo visu-

alization of tumor-feeding vessels. NIR-II tumor blood vessel fluorescence images of the 4T1 breast

tumor-bearing mice obtained at 2 min post tail vein injection of the probe nanoparticles (0.2 mL, 10

mg kg−1) with (b) 1000 nm (200 ms, 808 nm excitation, 90 mW cm−2) and (c) 1250 nm (800 ms, 808 nm

excitation, 180 mW cm−2) LP filters, respectively. The red circles indicate the tumor location, and

yellow arrows indicate the tumor-feeding arteries. (d,e) NIR-II fluorescence images of the hindlimb

vessels in C57BL/6 mice at 2 min after tail vein injection of the probe nanoparticles (0.2 mL, 15 mg

kg−1) with (d) 1000 nm (60 ms, 808 nm excitation, 90 mW cm−2) and (e) 1250 nm (500 ms, 808 nm

excitation, 180 mW cm−2) LP filters, respectively. (f) In vivo NIR-II fluorescence images (1250 nm LP,

400 ms, 808 nm excitation, 180 mW cm−2) of incomplete left hindlimb ischemia pre-injection, and 60

s, 120 s, and 180 s after injection of the probe nanoparticles; the white arrow indicates the occlusion

site in the femoral artery. (g) Intravital long-term hindlimb vasculature NIR-II imaging (1250 nm

LP, 400 ms, 808 nm excitation, 180 mW cm−2) from 0 min to 240 min after tail vein injection of the

probe nanoparticles. Scale bar (b–g): 1 cm. (h) The vessel FWHM width based on the cross-sectional

intensity profile measured along the white line in (g) (1 min) with the peak fitted to Gaussian func-

tions (the red curve is the Gaussian fit to the profile). Reprinted with permission from Ref. [112]. Copyright 2019, Royal Society of Chemistry.

Int. J. Mol. Sci. 2022, 23, 4949 26 of 52

4. Polymer Probes for Bioimaging

Table 4 introduces key chemical units that have been frequently used in the design

of polymeric luminescent probes. Benzothiadiazole (BTD) is a heteroaromatic compound

containing S and N atoms. BTD derivatives, including BBTD, with photophysical and elec-

tron-withdrawing properties have potential applications in organic light-emitting diodes,

photovoltaic cells, and organic field-effect transistors [198,202,235,236]. Pentiptycene is a

member of the iptycene family characterized by five benzene rings fused to a rigid H-

shaped scaffold with more than three times the internal free volume of the triptycene unit

[237]. The structure of pentiphene has been widely studied in microporous organic poly-

mers, MOFs, and COFs due its great potential to increase microporosity and molecular

adsorption capacity. Triazoles with a 5-membered ring of two carbon atoms and three

nitrogen atoms can be divided into 1,2,3-triazole and 1,2,4-triazole and are easily obtained

[238]. Triazole derivatives can act as bioisosteres of amides, esters, and carboxylic acids.

Therefore, triazole derivatives have various pharmacological properties such as antibac-

terial, anticancer, and anti-tuberculosis activities. Moreover, their solubility and binding

strength can be improved through various non-covalent interactions between the deriva-

tive and the biomolecule target. Fluorene, an ortho-fused tricyclic hydrocarbon, has a vi-

olet fluorescence [239]. Fluorene and its derivatives have been widely used as key materi-

als in various biomedical and optoelectronic applications. The fluorene unit is frequently

used to enhance molecular rigidity and reduce the intermolecular interaction in molecular

probes. Spirobifluorene comprises two fluorene units connected through the spiro-carbon

atom [240,241]. This unique spatial configuration induces strong rigidity with enhanced

stability, preventing the oxidation process occurring at position 9 of the fluorene ring.

Similarly to fluorene, spirobifluorene can also be chemically modified with donor and/or

acceptor groups to tune the emission of the system.

Quinoxaline, also called benzopyrazine, is a well-known nitrogen-containing heter-

ocyclic compound composed of benzene and pyrazine rings [242,243]. The quinoxaline

unit, which contains electron-withdrawing nitrogen atoms, is highly electron-deficient

and, thus, may serve as an efficient electron acceptor. Boron-dipyrromethene (BODIPY) is

an important class of versatile fluorophores with remarkable optical properties [244–246].

The use of the BODIPY unit may offer several advantages such as (i) strong absorption

and photoluminescence throughout the UV–vis region that can be tuned toward the NIR

region; (ii) a relatively high molar extinction coefficient and photoluminescence quantum

yield; (iii) photochemical and thermal robustness, high electron affinity, and ease of syn-

thetic functionalization; and (iv) tunable electronic and photonic properties.

Porphyrins are a class of macrocycles consisting of four pyrrole rings conjugated

through methine bridges [247,248]. Due to their large π-aromatic systems, porphyrins

possess excellent chemical and thermal stability as well as distinct photophysical and elec-

trochemical properties, which can be regulated by substituting the functional groups and

the coordinated metal ions. In addition to the coordination of metal ions at the porphyrin

center, the periphery of porphyrins can also be bound to metal ions if the peripheral rings

are properly functionalized. Intricate binding modes enable porphyrins to form desirable

molecular cages or a solid framework. The ability of porphyrins and their derivatives to

strongly absorb light at wavelengths near 400 nm has been widely exploited for various

light-harvesting applications. Lastly, methylene blue, a blue cationic thiazine dye, is

widely used in various industrial areas such as in food, cosmetics, textiles, pharmaceuti-

cals, and medicine [249,250]. Remarkably, methylene blue has received FDA approval for

the treatment of methemoglobinemia. Methylene blue has an absorption peak at 665 nm

with a shoulder at 610 nm and an emission at ~690 nm.

Int. J. Mol. Sci. 2022, 23, 4949 27 of 52

Table 4. Summary of the chemical units of polymer-based photoluminescence probes a.

Full Name

(Abbreviation)

Chemical

Structure Characteristics Application

2,1,3-Benzothiadia-

zole

(BTD)

- Strong electron-withdrawing capacity

- Candidate electron carriers

- Highly polarized property

- Induces intermolecular interactions

- Fungicides

- Herbicides

- Antibacterials

- Plant growth and protection

- Gene regulation

Pentiptycene

- Structural rigidity

- Nonplanarity

- Bulkiness

- π-electron richness

- Microporous materials

- Host–guest chemistry

- Membranes

Triazole

- Strong dipole moment

- π-electron-deficient aromaticity

- Hydrogen bond-accepting properties

- Good metabolic stability

- Bioisosteres

- Antibacterial/antifungal agents

- Anticancer agents

Fluorene

- Rigid planar structure

- High photoluminescence efficiency

- Good thermal stability

- Low cytotoxicity

- OLEDs b

- TADF c emitter

Spirobifluorene

- Structural rigidity

- Good thermal stability

- OLEDs

- Organic photovoltaics

Quinoxaline

- Good charge-transporting properties

- Dopant-free hole transporting materials

- Various derivatives

- Dyes

- Pharmaceuticals

- Antibiotics

Boron-dipyrrome-

thene

(BODIPY)

- High quantum yield

- Photochemical and thermal robustness

- High solubility in organic solvents

- Ease of synthetic functionalization

-High molar extinction coefficient and fluorescence

quantum yield

- Sensors

- Optoelectronic devices

- Therapeutic application

- Redox flow batteries

Porphyrin HN

N

NH

N

- Absorb light at wavelengths near 400 nm

- Ability to coordinate with metal ions

- Facile derivatization

- Supramolecular self-assembly

- Light-harvesting applications

- Biomimetic catalysis

- Photodynamic therapy

Methylene blue

- High water solubility


- Good charge delocalization

- Relatively low hydrogen bond donor capabilities

- Staining agent

- Pharmaceutical agent

- Polyelectrolytes

- Nitrogen-based ligands

- Photoelectronic redox-active

chemistries a Note that the toxicity of a unit cannot be directly associated with that of fluorescent probes based

on the unit; b OLED, organic light-emitting diode; c TADF: thermally activated delayed fluorescence.

4.1. UV/NIR Polymer Probes

Figures 14–16 summarize the molecular structures of photoluminescent Pdots that

have been employed for bioimaging in recent studies. Figures 14 and 15 present single-

polymer probes, and Figure 16 shows examples of mixing two or more molecules at an

appropriate ratio to prepare multi-component polymer probes. Table 5 summarizes the

Int. J. Mol. Sci. 2022, 23, 4949 28 of 52

excitation peak, emission peak, and Stokes shifts of reported polymer probes. To date,

there have been only limited examples of acquiring polymer probes with both absorption

and emission maxima in the NIR-II window. Figure 17 presents a good example of the

design of polymer probes to obtain desirable photophysical properties. NIR-II probes 24–

26 are semiconducting polymers with a bulky architecture to provide anti-ACQ properties

[8]. Both the rigid three-dimensional Pttc segment and the bulky SeBTa segment may pro-

vide steric hindrance to prevent the ACQ phenomenon of post-linked NIR-II fluorene-

based monomers (fluorene derivatives with alkyne-functionalized polymethine-cyanine

dyes). The covalent binding of the polymers with the NIR-II fluorophores is an efficient

strategy to circumvent the fluorophore diffusion/leakage encountered by small-molecule

fluorophore-entrapped/encapsulated polymeric nanoparticles. It is important to note that

the fluorophore leaching problem may cause inconsistent optical signals and potential

cytotoxicity, particularly for in vivo imaging applications. The Pttc and polyfluorene seg-

ments resulted in an absorption peak at ~450 nm and the SeBTa segment had an absorp-

tion peak at ~820 nm, while the polymethine-cyanine monomers were responsible for

broad absorption ranging from 900 to 1400 nm. This indicates that probes 24–26 can be

readily excited by long-wavelength laser sources depending on the measurement condi-

tion required for specific applications.

Int. J. Mol. Sci. 2022, 23, 4949 29 of 52

Figure 14. Chemical structures of the polymer probes constituting polymer dots, via the strategy of

tuning the alkyl structure to change their optical properties.

Int. J. Mol. Sci. 2022, 23, 4949 30 of 52

Figure 15. Chemical structures of the polymer probes constituting polymer dots.

Int. J. Mol. Sci. 2022, 23, 4949 31 of 52

Figure 16. Chemical structures of the polymer probes constituting polymer dots when over two

molecules are used.

Int. J. Mol. Sci. 2022, 23, 4949 32 of 52

Table 5. Summary of the optical properties of photoluminescence polymer dots (Pdots) used for

bioimaging.

Material Pdots λex

(nm)

Pdots λem

(nm)

Stokes Shifts

(nm) Ref.

24 936, 1066 (Probe)

400, 820, 1082 (Pdots)

1125 (Probe)

1115 (Pdots)

59 (Probe)

33 (Pdots)

[8] 25 1050, 1188 (Probe)

400, 820, 1082 (Pdots)

1270 (Probe)

1225 (Pdots)

82 (Probe)

143 (Pdots)

26 1140, 1270 (Probe)

400, 820, 1200 (Pdots)

1380 (Probe)

1300 (Pdots)

110 (Probe)

100 (Pdots)

27 497 631 134

[49]

28 497 603 106

29 501 659 158

30 520 682 162

31 593 653 60

32 477, 560 710 150

33 622 733 111

34 835 1035 200

35 736 1040 304

36 812 934 122

37 354, 600 668 68

[121]

38 338, 574 618 44

39 319, 574 592 18

40 538 600 62

41 387, 498 571 73

42 392, 456 554 98

43 452 475 23

[118] 44 452 476 24

45 449 474 25

46 390 515 125 [119]

47 528 550 22 [122]

48 445, 600 725 125 [251]

49 470 645 175 [117]

50 -- 651, 713 -- [120]

51 570 630 60 [252]

52 570 -- --

Int. J. Mol. Sci. 2022, 23, 4949 33 of 52

Figure 17. (a) Chemical structures of polymethine dye-conjugated semiconducting polymers. Three

types of NIR-II fluorescent polymethine dyes (NIR1125, NIR1270, and NIR1380) were synthesized

with activable alkyne functional groups for further conjugation with the semiconducting polymer.

(b) Absorption spectra of NIR1125 (red line), NIR1270 (green line), and NIR1380 (blue line) in

CH2Cl2. The middle-left inset shows a photograph of the dye solutions. (c) Emission spectra of

NIR1125 (red line), NIR1270 (green line), and NIR1380 (blue line) in CH2Cl2. (d) Synthesis of a sem-

iconducting polymer bearing carboxylic acid groups for further transformation to azide groups, fol-

lowed by CuAAC click reactions with polymethine dyes. Reprinted with permission from Ref. [8].

Copyright 2020, John Wiley and Sons.

As another example, Habuchi and colleagues [49] synthesized probes 27–36 using a

conjugated polymer with a bent and twisted structure to suppress the generation of fluo-

rescence quenching. Specifically, probes 27–36 comprised an NIR-emitting polycarbazole-

based donor-acceptor-type conjugated polymer and a polyfluorene-based planar conju-

gated polymer. The fluorescence brightness of the polymer probes was found to depend

on (i) fluorescence quenching inside the polymer chains and (ii) intramolecular twisting

between the donor and acceptor moieties of the polymer. These findings provide im-

portant insight, at the molecular level, into the development of NIR polymer probes with

a high quantum yield.

Int. J. Mol. Sci. 2022, 23, 4949 34 of 52

4.2. Nanoparticulate Polymer Probes

To date, the most common method for preparing nanoparticulate polymer probes

(mostly referred to as Pdots) has been the nanoprecipitation method. The preparation of

NIR-II-probe 24–26-based Pdots with surface carboxyl groups, illustrated in Figure 18a,

followed the traditional nanoprecipitation method by co-precipitating amphiphilic lipids

into the Pdots. The probe 25 Pdots were optically unstable in aqueous solutions, especially

under light excitation, whereas they were stable in most organic solvents. By contrast,

probe 24 and 26 Pdots were highly stable in organic solvents. The resulting Pdots showed

hydrodynamic diameters of 35–73 nm. Figure 18b shows the particle size distribution of

probe 31 Pdots along with their corresponding TEM images.

Figure 18. (a) Schematic showing the preparation of lipid-protected polymer dots (Pdots) for in vivo

bioimaging. (b) Representative hydrodynamic sizes of probe 24 Pdots measured by dynamic light

scattering. The upper-right inset represents the corresponding transmission electron microscopy

image with a scale bar of 50 nm. Reprinted with permission from Ref. [8]. Copyright 2020, John

Wiley and Sons.

The size uniformity of Pdots is also an important issue for bioimaging applications

[253–256]. Recently, a few studies have confirmed the successful preparation of uniform-

sized Pdots. Chochos and colleagues [121] reported the preparation of NIR Pdots, consist-

ing of probes 37–42, via both nanoprecipitation and encapsulation methods, as illustrated

in Figure 19. For nanoprecipitation, each polymer was dissolved in THF, which is an apro-

tic polar solvent with a low boiling point, but is miscible with water. Subsequently, the

polymer THF solution was added dropwise into water under sonication. The THF is read-

ily evaporated even at room temperature, resulting in solidified nanoparticles. However,

for encapsulation, the polymer was dissolved with an amphiphilic block copolymer,

poly(ethylene glycol)methyl ether-block-poly(lactide-co-glycolide) (mPEG-b-PLGA), in

THF. Note that mPEG-b-PLGA is an FDA-approved and metabolizable polymer. The rest

of the procedure was the same as that described for the nanoprecipitation method. Stable

aqueous-phase Pdots were then obtained through aggregate formation via hydrophobic

interactions between the polymer chains. In the case of encapsulation, the hydrophobic

PLGA chains would be liable to entangle with the polymer chains, while the hydrophilic

PEG chains would extend into the aqueous phase. The obtained conjugated polymer na-

noparticles (CPNs) were then filtered through a 0.2 μm cellulose acetate filter. The result-

ing concentrations (before filtration) of CPNs prepared via the nanoprecipitation method

Int. J. Mol. Sci. 2022, 23, 4949 35 of 52

were 2.8 ppm for the TQ polymer and 4.3 ppm for all the others, but it was 143 ppm for

CPNs prepared via the encapsulation method. TEM observation revealed a distinguisha-

ble, approximately spherical shape for all nanoparticles (Figure 19b). The diameters of the

nanoparticles ranged from 24 nm to 40 nm for the nanoprecipitation method and from 17

nm to 20 nm for the encapsulation method. These average particle sizes, less than 50 nm,

indicate the availability of Pdots for various biological applications [257–259].

As shown in Figure 20, probe 48 was designed to include strong electron-donating and

electron-deficient units into the polymer backbone, which resulted in strong ICT emission

at long wavelengths. The π-stacking between the polymer backbones was minimized by

adding bulky 4-(octyloxy)phenyl groups, which would prevent undesirable intermolecular

aggregation to yield highly efficient fluorescence inside the nanoparticles. Probe 48 was only

soluble in organic solvents such as THF and toluene. Therefore, for biological applications,

probe 48 was formulated into Pdots with the aid of an amphiphilic copolymer, 1,2-dis-

tearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-

PEG), using a nanoprecipitation method (Figure 20a). Typically, a THF solution containing

dissolved probe 48 and DSPE-PEG was quickly injected into water, and then this mixture

solution was subject to continuous probe sonication. The hydrophobic polymer would ag-

gregate into small nanoparticles with the hydrophobic segment of DSPE-PEG in aqueous

solution, while the hydrophilic PEG chains would extend to the aqueous phase. Im-

portantly, the hydrodynamic size of the resulting Pdots depended on the amount of DSPE-

PEG used. The hydrodynamic sizes of the Pdots were 4 nm (probe 48 Pdot1) and 34 nm

(probe 48 Pdot2) at DSPE-PEG-to-probe 48 weight ratios of 10:1 and 2:1 ratio, respectively

(Figure 20b,c). The zeta potentials of the Pdots were measured to be approximately −45 to

−51 mV, indicating that they have good colloidal stability in the aqueous phase.

Figure 19. (a) Schematic representation of the conjugated polymer nanoparticles prepared through

nanoprecipitation (top) and encapsulation (bottom). (b,c) Photographs that confirm the morphology

by manufacturing polymer dots (Pdots) with a photoluminescence polymer. (b) Size distribution of

Int. J. Mol. Sci. 2022, 23, 4949 36 of 52

aqueous probe 37–42 nanoparticles prepared via the nanoprecipitation method determined by dy-

namic light scattering (DLS) measurements and their morphology determined using transmission

electron microscopy (TEM). (c) Size distribution of aqueous probe 37–42 Pdots prepared via the

encapsulation method determined by DLS measurements and their morphology determined using

TEM. Reprinted with permission from Ref. [121]. Copyright 2020, Royal Society of Chemistry.

Figure 20. (a) Chemical structures of probe 48 and DSPE-PEG polymers and a schematic illustration

for probe 48 Pdots synthesis. Hydrodynamic size distributions of DSPE-PEG:probe 48 weight ratios of

(b) 10:1 and (c) 2:1 in aqueous media. The insets are the corresponding TEM images of 10:1 and 2:1.

Reprinted with permission from Ref. [251]. Copyright 2019, Wiley-VCH Verlag GmBH & Co. KGAA.

4.3. In Vivo Bioimaging

As one of the most recent studies on Pdots-based in vivo bioimaging, Chan and col-

leagues [8] reported the synthesis of probe 24 and probe 26 Pdots for in vivo non-invasive

fluorescence imaging in mice. Prior to in vivo applications, they performed MTT assays

to evaluate the cytotoxicity of Pdots at different concentrations, which confirmed that

Pdots have only minimal cell toxicity. As shown in Figure 21a, irradiation was performed

with an 808 nm laser source and low excitation power of 5 mW cm−2 to minimize photo-

damage to mice as well as the Pdots, which was equipped with different LP filters (1020,

1100, 1250, and 1312 nm). The use of a shorter LP filter (i.e., 1020 nm) was advantageous

to achieve stronger fluorescence. However, the spatial resolution and the SBR for in vivo

imaging showed the opposite trend due to strong background interference. By consider-

ing this result, a 1100 nm LP filter and a 1250 nm LP filter were used for probe 24 Pdots

(traditional NIR-II) and probe 26 Pdots (NIR-IIb), respectively.

Whole-body imaging in live mice was then performed by intravenously injecting the

two Pdots separately into mice via the tail vein and comparing their performance (Figure

21b–e). The excitation power laser density was 5 mW cm−2 with an exposure time of 800

ms. The mice were intravenously injected through the tail vein with 200 μL of Pdots (1.6

mg mL−1) or ICG (200 μg mL−1). NIR-IIb imaging was performed at 10 min post-injection.

For ICG control, all experimental conditions, including the laser power and filter sets,

were the same as those used in the experiments with probe 26 Pdots, except that the ac-

quisition time was 5 min post-injection due to the fast renal clearance rate of ICG.

Int. J. Mol. Sci. 2022, 23, 4949 37 of 52

Fluorescence imaging performed by using probe 26 Pdots equipped with a 1250 nm LP

filter revealed a significantly higher SBR than found with the 1125 nm LP filter (Figure

21b,c). By contrast, the imaging resolution obtained by ICG appeared to be very poor, as

presented in Figure 21d. The two Pdots showed a similar spatial resolution of 0.53–0.61

mm (Figure 21e), whereas the SBR of probe 26 Pdots (SBR ~2.3) was approximately 1.2-

times higher than that of probe 24 Pdots (Figure 21f). Importantly, the above results

demonstrated the advantages of NIR-IIb imaging with nearly zero autofluorescence and

minimal photon scattering. In addition, as shown in Figure 21g,h, the blood vessels close

to the spinal cord were also readily visualized, demonstrating that probe 26 Pdots ren-

dered a higher resolution due to low background interference. The cerebral blood vessels

of the mouse through the intact scalp and skull were distinctly visualized along with the

delicate vascular structures (Figure 21i). The high resolution of NIR-IIb imaging was again

highlighted when using probe 26 Pdots (middle panel in Figure 21i). The purpose of this

study was to diagnose the malignant brain tumor in vivo among the vascular morphology

in the mouse brain, using the ND2:SmoA1 transgenic mouse as the model. As shown in

Figure 21j, the brain vasculature in the ND2:SmoA1 mouse exhibited a major area of struc-

tural disorder in the brain vasculature. By contrast, the brain vasculature in the wild-type

C57BL/6 mouse showed an orderly arrangement and distribution in the brain. The mice

were further anatomized to analyze the distribution of the Pdots in major organs, where

fluorescence was found mostly in the liver, kidney, and spleen (Figure 21k). The high ac-

cumulation of Pdots in the liver and spleen indicated that the hepatobiliary clearance sys-

tem was the main metabolic pathway of Pdots. These results demonstrated that Pdots are

suitable for deep-tissue NIR-II imaging, enabling the early diagnosis of diseases associ-

ated with a brain blood vessel abnormality.

Another non-invasive brain imaging method was also developed using probe 48

Pdots synthesized by the Liu group [251] (Figure 22). Two-photon fluorescence (2PF) mi-

croscopy with NIR light excitation was used as a non-invasive tool to examine the brain

with a high spatial resolution and large imaging depth. Figure 22a demonstrates the deep

in vivo 2PF imaging of the intact mouse brain using probe 48 Pdots, which are excitable

by NIR-II light. Two Pdots of different size, namely probe 48 Pdot1 (~4 nm) and probe 48

Pdot2 (~34 nm), were prepared using the method described in Figure 20. The absorption

and emission spectra of the probe 48 Pdots in aqueous solution are plotted in Figure 22b

and 22c. The suspensions of probe 48 Pdot1 and probe 48 Pdot2 had similar absorption

spectra with two peaks centered at approximately 445 nm and 600 nm. The photolumi-

nescence spectra of both probe 48 Pdots exhibited a broad emission from 650 nm to 1000

nm. However, the fluorescence intensity of probe 48 Pdot1 was 4-times higher than that

of probe 48 Pdot2 in aqueous media at the same concentration. The 2PF microscopy ob-

servation of 48 Pdots-labeled blood vessels in the same mouse brain with 800, 1040, and

1200 nm excitation indicated that longer-wavelength excitation reduced the background

noise and improved the SBR of images. In a mouse model with a cranial window, in vivo

2PF imaging of probe 48 Pdots-stained brain blood vessels was achieved with an SBR of

~6 at a depth of 1010 μm for under 1200 nm femtosecond laser excitation. Through-skull

2PFM effectively visualized the blood vasculature architectures in the bone marrow

within and beneath the skull. Importantly, a three-dimensional reconstruction of 2PF im-

ages of the brain vasculature network with high contrast and a large vertical depth of 400

μm through the intact skull was also successfully obtained (Figure 22d–o).

The Wu group [120] reported the development of probe 50 Pdots for non-invasive in

vivo hypoxia fluorescence imaging in mice. Probe 50 consisted of a hydrophobic polymer

(PFPtTFPP) as an oxygen sensor with fluorene as a donor and Pt(II) porphyrin decorated

with fluorobenzene as an acceptor (Figure 23a). As represented in Figure 23b,c, probe 50

Pdots with a size of approximately 21 nm were prepared using nanoprecipitation with

poly(styrene-co-maleic anhydride). Blue and red emissions were observed from the Pdots

under 374 nm excitation in an O2 atmosphere due to the fluorene moieties and Pt(II) por-

phyrin, respectively (Figure 23d). The capability of probe 50 Pdots for hypoxia imaging

Int. J. Mol. Sci. 2022, 23, 4949 38 of 52

was first evaluated in vitro with MCF-7 cancer cells (Figure 23e). Initially, almost no phos-

phorescence was detected under a 5% CO2 in air atmosphere. Subsequently, a cover glass

with 2 mm thickness and 10 mm diameter was placed on the cells for 1 h; the cover glass

decreased the oxygen supply, thus generating hypoxia at the center of the glass. Fluores-

cence microscopy demonstrated that phosphorescence intensity significantly increased.

After the cover glass was removed and the cells were incubated for another 10 min under

5 % CO2 in air, the phosphorescence intensity recovered to its initial weak state. Cycles of

hypoxia–normoxia produced by repeatedly placing and removing the cover glass resulted

in the same phosphorescence intensity changes over another two cycles. These results

demonstrated that the probe 50 Pdots can present high reversibility for detecting repeated

hypoxia–normoxia cycles. Next, hypoxia imaging in vivo was performed with subcutane-

ously tumor-implanted nude mice. The mice were intratumorally and subcutaneously in-

jected with a probe 50 Pdots solution (10 μL, 1.6 mg mL−1), respectively. Whole-body im-

aging showed that the fluorescence intensity in the tumor area (region of interest (ROI) 2)

was much higher than that of the subcutaneous injection area (ROI 1) (Figure 23f,g). This

is mainly because the oxygen concentration in the tumor area is close to 0% and, thus,

lower than that in the subcutaneous area. Collectively, these results indicated that probe

50 Pdots are highly sensitive to oxygen and, thus, have strong potential for hypoxia im-

aging in vivo.

Figure 21. (a) Photographs of probe 24 Pdots (left) and probe 26 Pdots (right) under ambient light

(first panel) and under 808 nm laser excitation with a 1020 nm LP filter (second panel), 1100 nm LP

filter (third panel), 1250 nm LP filter (fourth panel), and 1312 nm LP filter (fifth panel). (b) Whole-

body imaging of live mice intravenously injected with probe 24 Pdots in supine (left) and prone

(right) positions with a 1100 nm LP filter. (c) Whole-body imaging of live mice intravenously in-

jected with probe 26 Pdots in supine (left) and prone (right) positions with a 1250 nm LP filter. (d)

Whole-body imaging of live mice intravenously injected with ICG in supine (left) and prone (right)

positions with a 1250 nm LP filter. (e) Enlarged view of the mouse hindlimb vasculature in the green

squares of (b) (upper panel) and (c) (bottom panel), respectively. (f) Cross-sectional intensity pro-

files along the green lines in (e) for probe 24 Pdots (left) and probe 26 Pdots (right), respectively. (g)

Enlarged view of the area close to the spinal cord in the red squares of (b) (upper panel) and (c)

(bottom panel), respectively. (h) Cross-sectional intensity profiles along the red lines in (g) for probe

24 Pdots (left) and probe 26 Pdots (right), respectively. (i) NIR-II fluorescence imaging of the wild-

Int. J. Mol. Sci. 2022, 23, 4949 39 of 52

type C57BL/6 mouse brain vasculature after intravenous injection of probe 24 Pdots (left, 1100 nm

LP filter), probe 26 Pdots (middle, 1250 nm LP filter), and ICG (right, 1250 nm LP filter). (j) NIR-II

fluorescence imaging of the ND2:SmoA1 mouse brain vasculature after intravenous injection of

probe 26 Pdots (1250 nm LP filter). (k) Accumulation of probe 24 Pdots (upper panel in the inset)

and probe 26 Pdots (bottom panel in the inset) in the major excised organs at 24 h post-injection.

Their corresponding quantitative mean fluorescence intensities are also plotted. The scale bars are

10 mm, 10 mm, and 5 mm in (b), (c), and (i), respectively. Reprinted with permission from Ref. [8].

Copyright 2020, John Wiley and Sons.

Figure 22. (a) Schematic illustration of NIR-I and NIR-II excited with in vivo 2PF imaging of the

mouse brain. (b) Normalized UV–vis absorption spectra of probe 48 Pdots in aqueous media; inset

is the digital photograph of the two probe 48 Pdots in cuvettes. (c) Photoluminescence spectra of

probe 48 Pdots at a concentration of 10 μg mL−1 in aqueous solution; the inset shows a digital pho-

tograph of the two probe 48 Pdots in cuvettes under a UV lamp. In vivo 2PF imaging of probe 48

Pdot-stained mouse brain blood vessels with an intact skull. (d,e) Three-dimensional reconstructed

second harmonic generation (SHG) images of the mouse skull. (f–k) 2PF images of probe 48 Pdot-

stained brain blood vessels at various vertical depths. (l–n) Three-dimensional reconstructed 2PF

images of the brain blood vessels. (o) Three-dimensional reconstructed image showing the position

of the skull and the brain blood vessels network below the skull. Excitation for SHG: 950 nm; exci-

tation for 2PF: 1200 nm. Emissions were collected within 455–500 nm for SHG and 660–750 nm for

CPdots1. Each frame was acquired in 3.22 s. The length of scale bars in all images is 100 μm. Re-

printed with permission from Ref. [251]. Copyright 2019, John Wiley and Sons.

Int. J. Mol. Sci. 2022, 23, 4949 40 of 52

Figure 23. (a) Synthetic routes of polymer PFPtTFPP and (b) preparation of corresponding probe 50

Pdots. (c) Size distribution of probe 50 Pdots in aqueous solution measured by dynamic light scat-

tering (DLS). (d) Emission spectra of probe 50 Pdots under different dissolved oxygen. Inset photo-

graphs show the oxygen and nitrogen atmosphere under 365 nm illumination. (e) Fluorescence im-

aging of MCF-7 cells without (1, 3, 5) and with (2, 4, 6) a cover glass. The top to bottom show fluo-

rescence images, bright-field images, and combined bright-field and fluorescence images. The scale

bar represents 75 μm. (f) Whole-body imaging of mice after subcutaneous (ROI 1) and intratumoral

(ROI 2) injection of 1.6 mg mL−1 probe 50 Pdots solution. (g) Semiquantitative analysis of brightness

in ROI 1 and ROI 2 areas. Reprinted with permission from Ref. [120]. Copyright 2019, Wiley-VCH

Verlag GmBH & Co. KGAA.

Int. J. Mol. Sci. 2022, 23, 4949 41 of 52

5. Conclusions

To date, various efforts have been devoted to developing novel bioimaging tech-

niques for practical applications. Photoluminescent probes such as small molecules,

Pdots, CDs, graphene dots, MOFs, and COFs are promising candidates due to their inter-

esting properties. This review illustrates the recent advances in the bioimaging strategies

using photoluminescent small molecules and Pdots for in vivo applications. There is no

doubt that these probes show excellent photoluminescent performance that can be uti-

lized for in vitro and in vivo imaging applications. We have highlighted some of the re-

cently explored methods and strategies for the preparation, evaluation, and application

of nanoparticulate photoluminescent probes. Small-molecule probes for bioimaging have

shown limitations owing to their low water dispersion (dissolution) and lack of photosta-

bility. Recent studies using small molecules for bioimaging have tried to overcome these

drawbacks by shaping them into nanoparticles, frequently with appropriate biocompati-

ble amphiphiles. Moreover, the use of polymers has some common advantages such as

good biocompatibility, low toxicity, a tunable structure for the biotarget, and ease of de-

signing the NIR emission-range structure for enhancing their penetration to detect bi-

omarkers. Pdots have further advantages of good photostability, good water dispersion,

and easy functionalization.

In summary, this review has highlighted cases in which recently published organic

molecular probes, including small molecules and polymers, were successfully used for

bioimaging, along with the strategies for designing their structures, their strengths and

weaknesses, and actual bioimaging effects. The importance of the bioimaging field con-

tinues to grow annually. In particular, due to global pandemics such as COVID-19, the

development of bioimaging technology is increasingly necessary to easily detect and di-

agnose diseases, which will lead to the emergence of new types of bioimaging fields.

We hope that this review will help in the design of probes for future bioimaging research.

Author Contributions: Conceptualization, S.L., C.S.P. and H.Y.; writing—original draft preparation,

S.L., C.S.P. and H.Y.; writing—review and editing, S.L., C.S.P. and H.Y.; supervision, H.Y..; funding ac-

quisition, H.Y. All authors have read and agreed to the published version of the manuscript.

Funding: This research was supported by the Basic Science Research Program through the National

Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-

2020R1I1A3072666).

Acknowledgments: H.Y. acknowledges the support of the NRF of Korea.

Conflicts of Interest: The authors declare no conflict of interest.

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